Novel-anti-infective strategy against influenza virus and s. aureus coinfections

ABSTRACT

The present invention relates to MEK inhibitor, p38 inhibitor and/or NFκB inhibitor for use in a method for the prophylaxis and/or treatment of a co-infection comprising a bacterial infection and an influenza virus infection or a bacterial infection alone. Also provided are compositions comprising such inhibitors for use in the prophylaxis and/or treatment of a co-infection comprising a bacterial infection and an influenza virus infection or a bacterial infection alone. In addition an in vitro test system, wherein the test system comprises cultured cells infected with an influenza virus and a bacterium or with a bacterium alone is provided.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/571,550, filed Sep. 16, 2019, which is a continuation of U.S.application Ser. No. 15/310,838, filed Nov. 14, 2016, now U.S. Pat. No.10,458,440 which claims priority to International Application No.PCT/IB2015/053644, filed May 18, 2015, which claims priority toLuxembourg Application No. 92455, filed May 16, 2014 wherein thecontents of said applications are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to MEK inhibit, p38 inhibitor and/or NFκBinhibitor for use in a method for the prophylaxis and/or treatment of aco-infection comprising a bacterial infection and an influenza virusinfection or a bacterial infection alone. Also provided are compositionscomprising such inhibitors for use in the prophylaxis and/or treatmentof a co-infection comprising a bacterial infection and an influenzavirus infection or a bacterial infection alone. In addition an in vitrotest system, wherein the test system comprises cultured cells infectedwith an influenza virus and a bacterium or with a bacterium alone isprovided.

BACKGROUND OF THE INVENTION

Influenza A viruses are the causative agents of severe respiratorydiseases resulting in significant morbidity and mortality. Most of thefatal cases in the course of an influenza virus (IV) infection areactually a result of secondary pneumonia caused by different bacteria,such as Staphylococcus aureus (S. aureus), Streptococcus pneumoniae andHaemophilus influenzae (Morens et al., 2008, Chertow et al., 2013). Themost striking problems of bacterial co-infection are the suddenlyincreased pathogenicity (Iwao et at., 2012, Paddock et al., 2012, Parkeret al., 2012) and a limited arsenal of potent anti-infectives againstthe different pathogens. The high variability of influenza viruses andthe continuous emergence of new strains (Neumann et al., 2009,Taubenberger et al., 2010, Parry, 2013), specific characteristics of thebacterial strains (Grundmann et al., 2006, Moran et al., 2008, Gillet etal., 2007, Shio et al., 2011), as well as the rapid resistancedevelopment of both, influenza viruses (Hayden et al., 1992, Bright etal., 2006, Pinto et al., 2006, De Clercq et al., 2007, Pinto et al.,2007) and bacteria (Grundmann et al., 2006, Moran et al., 2006, Shilo etal., 2011) against the available drugs/antibiotics are the major reasonsfor the poor treatment options Moreover, it is incidental that treatmentof coinfections with influenza viruses and bacteria is not possible witha single compound, so far. The current invention solves this problem inthat it proposes a novel anti-infective strategy against IV and S.aureus co-infections by using single drugs. Furthermore, the presentinvention solves the problem of rapid resistance development of bacteriaby providing drug that targets cellular factors rather than thebacterium itself.

SUMMARY OF THE INVENTION

The technical problem is solved by the embodiments reflected in theclaims, described in the description, and illustrated in the Examplesand Figures.

The above being said, the present invention relates to a MEK inhibitor,p38 inhibitor and/or NFκB inhibitor for use in a method for theprophylaxis and/or treatment of a co-infection comprising a bacterialinfection and an influenza virus infection.

In addition the present invention relates to a MEK inhibitor, p38inhibitor and/or NFκB inhibitor for use in a method for the prophylaxisand/or treatment of a bacterial infection.

Despite intensive research in the last century, IV still represent asevere threat to mankind. Seasonal outbreaks that are especiallydangerous for the elderly and immunocompromised individuals are due toinfections with influenza A or B viruses.

Within the last decades, there is an increasing incidence ofmethicillin-resistant S. aureus strains, causing problems especially ininfants and children who were concomitantly infected with IV (Iverson etal., 2011, Thorburn et al., 2012). One major problem occurring uponbacterial co-infections is the sudden and highly increasedpathogenicity, which is probably caused by accelerated cytokineexpression, also resulting in tissue damage. Particularly, uponco-infection with Panton-Valentine leukocidin (PVL)-expressing S. aureussevere lung epithelium damage is observed, due to uncontrolled releaseof proteases after PVL-mediated neutrophil killing (Gillet et al, 2007,Niemann et al., 2012). Bacterial co-infections usually occur within thefirst six days of an IV infection, resulting in even more fulminantillness, pneumonia and higher mortality (Iverson et al., 2011, Chertowet al, 2013). However in same cases bacterial co-infection comes up,when virus-infection already seems to be cleared. For treatment ofviral/bacterial co-infections only limited possibilities exist.

One promising antiviral strategy to fight influenza is based on the factthat IV, as intracellular pathogens, strongly depend on the cellularsignaling machinery (Gong et al., 2009, Ludwig, 2009). IV acquired theability to highjack cellular factors for its own purpose (Ludwig etal.,2003). Furthermore, IV are able to suppress the innate immuneresponse of their hosts. Given these dependencies, cellularvirus-supportive functions are most promising candidates for novelantiviral intervention (Ludwig et al, 2003, Ludwig, 2011, Planz, 2013).During the last years we and others identified the Raf/MEK/ERK mitogenickinase cascade (Pleschka et al., 2001, Ludwig et al., 2004, Olschlageret al., 2004, Marjuki et al., 2006, Ludwig, 2009, Droebner et al.,2011), the IKK/NFκB module (Pleschka et al., 2001, Wurzer et al., 2004,Marjuki et al., 2006, Mazur et al., 2007, Ludwig et al., 2008, Dudek etal., 2010, Droebner et al., 2011, Ehrhardt et al., 2013, Haasbach etal., 2013), the p38-(Borgeling et al., 2014) and also the PI3K-signaling(Ehrhardt et at., 2006, Ehrhardt et al., 2007a, Ehrhardt et al., 2007b,Ehrhardt et al., 2009, Eierhoff et al., 2010) pathways as suitabletargets for an anti-viral approach.

Targeting cellular rather than viral factors prevents the problem ofresistance because the pathogen cannot replace the missing cellularfunction. For several cellular factors chemical compounds are availableand although in an early stage, some of them have entered clinicaltesting or are even already licensed.

In contrast to IV replication, S. aureus division is host-cellindependent. Novel antibacterial alternatives do not target essentialgene products elaborated by the pathogen, but inhibit virulence factorsduring S. aureus infection without killing the bacterium or boostinghost immunity (Park et al., 2012). Other strategies prevent colonizationof S. aureus in the human host (Park et al., 2012). These compounds alsoexhibit a lower potential to induce resistance. Recently, there isaccumulating evidence that S. aureus also uses cellular signaling forits own benefits during infection (Oviedo-Boyso et al., 2011), but suchbacterial-supportive cellular factors have not yet been characterized astargets for antibacterial therapy in detail.

The present inventors surprisingly observed, that drugs againstintracellular signaling factors, such as NFκB, MEK or p38 MAP kinase,that were previously shown to possess anti influenza activity, alsoexhibit anti S. aureus activity and reduces both viral- and bacterialtiters in a coinfection scenario. Thus, these signaling inhibitors aremost promising candidates for the treatment of IV or S. aureusinfections alone, but, most importantly also against severe influenzaaccompanied with bacterial coinfection.

In one embodiment, the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection or bacterial infection of the presentinvention, wherein the the bacterial infection is mediated by abacterium selected from the group consisting of Staphylococcaceae,Streptococcaceae, Legionellaceae, Pseudomonadaceae, Chlamydiaceae,Mycoplasmataceae, Enterobacteriaceae, Pseudomonadales and/orPasteurellaceae.

In another embodiment the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection of the present invention, wherein theinfluenza virus infection is mediated by influenza A virus or influenzaB virus, preferably the influenza A virus is H1N1, H2N2, H3N2, H6N1,H7N7, H7N9, H9N2 H10N7, H10N8 or H5N1. In one embodiment, the influenzaA virus is H1N1. In other embodiments, the influenza A virus is H3N2,H5N1 and H7N9. In additional embodiments, the influenza A virus is H3N2,H5N1, H1N1 and H7N9.

In a further embodiment the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection or bacterial infection of the presentinvention, wherein the MEK inhibitor is selected from the groupconsisting of U0126, PLX-4032, AZD6244, AZD8330, AS-703026, GSK-1120212,RDEA-119, RO-5126766, RO-4987655, CI-1040, PD-0325901, GDC-0973,TAK-733, PD98059, ARRY-438162, PF-3644022 and PD184352, preferablyAZD8330, GSK-1120212, U0126, GDC-0973, CI-1040, PD0325901, ARRY438162,PF-3644022 and AZD6244, most preferably U0126, GDC-0973, CI-1040,AZD8330 and GSK-1120212.

In another embodiment the MFK inhibitor, p32 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection or bacterial infection of the presentinvention, wherein the p38 inhibitor is selected from the groupconsisting of SB202190, LY2228820, CAY10571, SB 203580, Tie2 KinaseInhibitor,2-(4-Chlorophenyl)-4-(fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one,CGH 2466, SB220025, Antibiotic LL Z1640-2, TAK 715, SB202190hydrochloride, SKF 86002, AMG548, CMPD-1, EO 1428, JX 401, ML 3403, RWJ67657, SB 202190, SB 203580, SB 203580 hydrochloride, SB 239063, SCIO469, SX 011, TAK 715, Pamapimod, Losmapimod (GW856553), Dilmapimod(SB681323), VX 702, VX 745, Doramapimod (BIRB 796), BMS-582949,ARRY-797, PH797804 preferably VX-702, SB202190, Pamapimod, losmapimod(GW856553), Dilmapimod (SB681323), Doramapimod (BIRB 796), BMS-582949,ARRY-797, PH797804 and SCIO-469.

In another embodiment the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection or bacterial infection of the presentinvention, wherein the NFκB inhibitor is selected from the groupconsisting of LASAG (also called LG-ASA), SC75741, MG 132, TPCA-1, PCTC,IMD 0354, Luteolin, Caffeic acid phenethyl ester, Cardamonin, PF 184,IKK 16, SC 514, Withaferin A, Arctigenin, Bay 11-7085, PSI, PR 39, Ro106-9920, Bay 11-7821, ML-130, Celastrol, Tanshinone IIA, HU 211,Gliotoxin, CID 2858522, Honokiol, Andrographolide, 10Z-Hymenialdisine,ACHP, Pristimerin, Sulfasalazine, ML 120B dihydrochloride, Amlexanox,9-Methyistreptimidone, N-Stearoyl phytosphingosine,2-(1,8-naphthyridin-2-yl)-Phenol, 5-Aminosalicylic acid, BAY 11-7085,Ethyl 3,4-Dihydroxycinnamate, Helanalin, NF-κB Activation Inhibitor 11,JSH-23, Glucocorticoid Receptor Modulator, CpdA, PPM-18, aspirin (ASA),Pyrrolidinedithiocarbamic acid ammonium salt, (R)-MG132, SC75741Rocaglamide, Sodium salicylate, QNZ, PS-1145, CAY10512, bortezomib,salsalate, resveratrol, deoxyspergualin, sulindac, thalidomide,AGRO-100, CHS 828 and/or Curcumin preferably, bortezomib, curcumin,aspirin (ASA), salsalate, resveratrol, sodium salicylate, LASAG (alsocalled LG-ASA), deoxyspergualin, sulindac, thalidomide, AGRO-100, CHS828 even more preferably SC75741, ASA and LASAG (also called LG-ASA) andmost preferably LASAG (also called LG-ASA).

In additional embodiments, the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection or bacterial infection of the presentinvention, wherein the MEK inhibitor is combined with another MEKinhibitor, the p38 inhibitor and/or the NFκB inhibitor; the p38inhibitor is combined with another p38 inhibitor, the MEK inhibitorand/or the NFκB inhibitor or the NFκB inhibitor is combined with anotherNFκB inhibitor, the p38 inhibitor and/or the MEK inhibitor.

In further embodiments, the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection of the present invention, wherein the MEKinhibitor, the p38 inhibitor and/or the NFκB inhibitor are combined withone or more inhibitors targeting the influenza virus and/or thebacterium. In one embodiment, the MEK inhibitor, the p38 inhibitorand/or the NFκB inhibitor is/are administered contemporaneously,previously or subsequently to the one or more inhibitors targeting theinfluenza virus and/or the bacterium. As such the MEK inhibitor, p38inhibitor and/or NFκB inhibitor can be combined with 1, 2, 3, 4, 5, 6,7, or 8 inhibitors targeting the influenza virus. Similarly, the MEKinhibitor, p38 inhibitor and/or NFκB inhibitor can be combined with 1,2, 3, 4, 5, 6, 7, or 8 inhibitors targeting the bacterium.

In one embodiment, the one or more inhibitors targeting the influenzavirus is a neuraminidase inhibitor, preferably oseltamivir phosphate,zanamivir, oseltamivir or peramivir.

In another embodiment, the one or more inhibitors targeting theinfluenza virus is a compound targeting an ion channel protein (M2),preferably amantadine and/or rimantadine. In further embodiments, theone or more inhibitors targeting the influenza virus is a compoundtargeting polymerase or endonuclease activity via interfering with acomponent of the viral polymerase complex, PB1, PB2, PA or NP,preferably NP blocker Nucleozin or polymerase inhibitor T-705.

In further embodiments, the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a bacterial infection of the present invention, wherein theMEK inhibitor, the p38 inhibitor and/or the NFκB inhibitor are combinedwith one or more inhibitors targeting the bacterium.

In another embodiment, the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection or bacterial infection of the presentinvention, the one or more inhibitor targeting the bacterium is anantibiotic, preferably Gentamicin, Rifampicin, Lysosthaphin,Erythromycin, Levofloxacin Vancomycin, Teicoplanin, Penicillin andOxacillin.

In additional embodiments, the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection or bacterial infection of the presentinvention is in a subject, preferably a vertebrate.

Also provided for by the present invention is a composition, comprisinga MEK inhibitor, a p38 inhibitor and/or a NFκB inhibitor for use in amethod for the prophylaxis and/or treatment of a co-infection comprisinga bacterial infection and an influenza virus infection. Preferably, thecomposition further comprises a carrier.

The present invention also relates to a composition, comprising a MEKinhibitor, a p38 inhibitor and/or a NFκB inhibitor for use in a methodfor the prophylaxis and/or treatment of a bacterial infection.Preferably, the composition further comprises a carrier.

Also provided for by the present invention is a composition, comprisinga MEK inhibitor, a p38 inhibitor and/or a NFκB inhibitor and one or moreinhibitors targeting the influenza virus and/or the bacterium for use ina method for the prophylaxis and/or treatment of a co-infectioncomprising a bacterial infection and an influenza virus infection.Preferably, the composition further comprises a carrier.

The present invention also relates to a composition, comprising a MEKinhibitor, a p38 inhibitor and/or a NFκB inhibitor and one or moreinhibitors targeting the bacterium for use in a method for theprophylaxis and/or treatment of a a bacterial infection, Preferably, thecomposition further comprises a carrier.

In further embodiments, the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection of the present invention, wherein the MEKinhibitor, the p38 inhibitor and/or the NFκB inhibitor reduces both theviral and bacterial infection, when contacting it/them with an in vitrotest system, wherein the test system comprises cultured cells infectedwith

-   -   a) an influenza virus and    -   b) a bacterium        when compared to the in vitro test system before the contacting.

In one embodiment, the reduction of the viral infection is a reductionin plaque forming units (pfu)/ml and the reduction in the bacterialinfection is a reduction in colony forming units (CFU)/ml.

In another embodiment, the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a bacterial infection of the present invention, wherein theMEK inhibitor, the p38 inhibitor and/or the NEκB inhibitor reduces thebacterial infection, when contacting it/them with an in vitro testsystem, wherein the test system comprises cultured cells infected with abacterium, when compared to the in vitro test system before thecontacting.

The present invention also relates to an in vitro test system, whereinthe test system comprises

-   -   cultured cells infected with    -   a) an influenza virus and    -   b) a bacterium.

The invention also provides for the use of the in vitro test system ofof the present invention for the determination of inhibitors effectivein reducing a coinfection comprising a bacterial infection and aninfluenza virus infection. In one embodiment, the reduction of the viralinfection is a reduction in plaque forming units (pfu)/ml and thereduction in the bacterial infection is a reduction in colony formingunits (CFU)/ml.

In addition the present invention relates to a method for detectingmolecules effective in the prophylaxis and/or treatment of aco-infection comprising a bacterial infection and an influenza virusinfection comprising contacting the in vitro test system of the presentinvention with a compound of interest, wherein the compound of interestreduces both the viral and bacterial infection, compared to the in vitrotest system before the contacting.

The present invention also provides for an in vitro test system, whereinthe in vitro test system comprises cultured cells infected with abacterium.

The present invention, in addition, relates to a use of the in vitrotest system of the present invention for the determination of inhibitorseffective in reducing a bacterial infection.

Furthermore, the present invention relates to the use of the in vitrotest systems of the present invention for the examination of innate hostcell responses, which optionally includes examination of the level ofsignal transduction, resulting cytokine and chemokine expression,induction of apoptosis and necrosis and/or redox hemostasis regulatinghealth and disease.

Also provided for by the present invention is a method for detectingmolecules effective in the prophylaxis and/or treatment a bacterialinfection comprising contacting the in vitro test system of the presentinvention with a compound of interest, wherein the compound of interestreduces the bacterial infection, compared to the in vitro test systembefore the contacting.

The present invention furthermore relates to a cultured cell infectedwith an influenza virus and a bacterium.

Also provided for is a cultured cell infected with a bacterium.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures show:

FIG. 1: Time scale of co-infection procedure. Cells were infected withIV for 30 min. Co-infection with S. aureus 6850 was conducted or cellswere mock-treated. Extracellular bacteria were lysed and removed byantibiotic treatment 3 h post bacterial infection. After a PBS wash,cells were supplemented with fresh medium (DMEM/INV) and incubated up to18 hrs of viral infection

FIG. 2: The MEK inhibitor U0126 reduces IV titers (A/Puerto Rico/8/34)and S. aureus load, even in a co-infection situation. Human lungepithelial cells were seeded in 6-well plates (8×10⁵ cells/well) in 2 mlDMEM (10% FCS). 16-20 hrs after seeding, cells were rinsed and incubatedwith PBS/BA [0.2% bovine serum albumin (BSA), 1 mM MgCl₂, 0.9 mM CaCl₂,100 U/ml penicillin, 0.1 mg/ml streptomycin] (500 μl per 6 well) orPBS/BA containing the influenza virus A/Puerto Rico/8/34 at amultiplicity of infection (MOI=0.1) at 37° C. After 30 min incubation,the virus dilution was aspirated, cells were rinsed with PBS andsupplemented with invasion medium DMEM/INV [1% human serum albumin, 25nmol/l HEPES] (2 ml per 6 well) with or without S. aureus 6850 (MOI=0.5)in presence of 50 μM U0126 or DMSO (solvent control). 3 hrs postbacterial infection cells were treated with antibiotics to removeextracellular bacteria. Therefore cells were rinsed with PBS andsubsequently incubated with DMEM/INVantibiotics [2 μg/ml lysostaphin(Sigma)] (1 ml per 6 well) for 20 min at 37° C. After an additional washwith PBS cells were supplemented with DMEM/INV containing 50 μM U0126 orDMSO and 0.333 μg/ml Trypsin (Invitrogen). After an incubation period offurther 14 hrs at 37° C. IV titers and intracellular bacteria weredetermined as described in (Hrincius et al., 2010, Tuchscherr et al.,2011). IV titers are depicted as plaque forming units (pfu)/ml (A, C)and S. aureus titers are depicted as colonie forming units (CFU)/ml (B,D). Data represent the means±SD of three independent experiments withtwo biological samples. Statistical significance was evaluated by atwo-tailed two sample t-test (*p<0.05; **p<0.01; ***p<0.001). (E) S.aureus in presence of 50 μM U0126 results in reduced bacterial titers 18hrs upon incubation in comparison to DMSO treated bacteria. A definedamount of S. aureus 6850 suspension culture was diluted in DMEM/INVsupplemented with 50 μM U0126 or DMSO and incubated at 37° C. for 18hrs. Bacteria were diluted and determined by serial dilution on agarplates. Data represent the means±SD of three independent experimentswith two biological samples. Statistical significance was evaluated by atwo-tailed two sample t-test (*p<0.05; **p<0.01; ***p<0.001).

FIG. 3: The MEK inhibitor U0126 reduces IV titers (A/FPV/Bratislava/79)and S. aureus load, even in a co-infection situation. Human lungepithelial cells were seeded in 6-well plates (8×10⁵cells/well) in 2 mlDMEM [10% FCS]. 16-20 hrs after seeding, cells were rinsed and incubatedwith PBS/BA [0.2% bovine serum albumin (BSA), 1 mM MgCl₂, 0.9 mM CaCl₂,100 U/ml penicillin, 0.1 mg/ml streptomycin] (500 μl per 6 well) orPBS/BA containing the Influenza virus A/FPV/Bratislava/79 at amultiplicity of infection (MOI=0.001) at 37° C. After 30 min incubation,the virus dilution was aspirated, cells were rinsed with PBS andsupplemented with invasion medium DMEM/INV [1% human serum albumin, 25nmol/l HEPES] (2 ml per 6 well) with or without S. aureus 6850 (MOI=0.5)in presence of 50 μM U0126 or DMSO (solvent control). 3 hrs postbacterial infection cells were treated with antibiotics to removeextracellular bacteria. Therefore cells were rinsed with PBS andsubsequently incubated with DMEM/INVantibiotics [2 μg/ml lysostaphin(Sigma)] (1 ml per 6 well) for 20 min at 37° C. After an additional washwith PBS cells were supplemented with DMEM/INV containing 50 μM U0126 orDMSO. After an incubation period of further 14 hrs at 37° C. IV titersand intracellular bacteria were determined as described in (Hrincius etal., 2010; Tuchscherr et al., 2011). IV titers are depicted as plaqueforming units (pfu)/ml (A, C) and S. aureus titers are depicted ascolonie forming units (CFU)/ml (B, D). Data represent the means±SD oftwo independent experiments with two biological samples. Statisticalsignificance was evaluated by a two-tailed two sample t-test (*p<0.05;**p<0.01; ***p<0.001).

FIG. 4: The p38 inhibitor SB202190 reduces IV titers and S. aureus load,even in a co-infection situation. Human lung epithelial cells wereseeded in 6-well plates (8×10⁵ cells/well) in 2 ml DMEM [10% FCS]. 16-20hrs after seeding, cells were rinsed and incubated with PBS/BA [0.2%bovine serum albumin (BSA), 1 mM MgCl₂, 0.9 mM CaCl₂, 100 U/mlpenicillin, 0.1 mg/ml streptomycin] (500 μl per 6 well) or PBS/BAcontaining the Influenza virus A/Puerto Rico/8/34 at a multiplicity ofinfection (MOI=0.1) at 37° C. After 30 min incubation, the virusdilution was aspirated, cells were rinsed with PBS and supplemented withInvasion medium DMEM/INV [1% human serum albumin, 25 nmol/l HEPES] (2 mlper 6 well) with or without S. aureus 6850 (MOI=0.5) in presence of 10μM SB202190 or DMSO (solvent control). 3 hrs post bacterial infectioncells were treated with antibiotics to remove extracellular bacteria.Therefore cells were rinsed with PBS and subsequently incubated withDMEM/INVantibiotics [2 μg/ml lysostaphin (Sigma)] (1 ml per 6 well) for20 min at 37° C. After an additional wash with PBS cells weresupplemented with DMEM/INV containing 10 μM SB202190 or DMSO and 0.333μg/ml Trypsin (Invitrogen). After an Incubation period of further 14 hrsat 37° C. IV titers and intracellular bacteria were determined asdescribed in (Hrincius et al., 2010; Tuchscherr et al., 2011). IV titersare depicted as plaque forming units (pfu)/ml (A, C) and S. aureustiters are depicted as colonie forming units (CFU)/ml (B, D). Datarepresent the means±SD of three independent experiments with twobiological samples. Statistical significance was evaluated by atwo-tailed two sample t-test (*p<0.05; **p<0.01; ***p<0.001).

FIG. 5: The NF-kappaB (NFκB) inhibitor LG-ASA reduces IV titers and S.aureus load, even in a co-infection situation. Human lung epithelialcells were seeded in 6-well plates (8×10⁵ cells/well) in 2 ml DMEM (10%FCS). 16-20 hrs after seeding, cells were rinsed and incubated withPBS/BA [0.2% bovine serum albumin (BSA), 1 mM MgCl₂, 0.9 mM CaCl₂, 100U/ml penicillin, 0.1 mg/ml streptomycin] (500 μl per 6 well) or PBS/BAcontaining the influenza virus A/Puerto Rico/8/34 at a multiplicity ofinfection (MOI=0.1) at 37° C. After 30 min incubation, the virusdilution was aspirated, cells were rinsed with PBS and supplemented withinvasion medium DMEM/INV [1% human serum albumin, 25 nmol/l HEPES] (2 mlper 6 well) with or without S. aureus 6850 (MOI=0.5) in presence of 5 mMLG-ASA. Water was used as solvent control. 3 hrs post bacterialinfection cells were treated with antibiotics to remove extracellularbacteria. Therefore cells were rinsed with PBS and subsequentlyincubated with DMEM/INVantibiotics [10% FBS, 2 μg/ml lysostaphin(Sigma)] (1 ml per 6 well) for 20 min at 37° C. After an additional washwith PBS cells were supplemented with DMEM/INV containing 5 mM LG-ASA orwater and 0.333 μg/ml Trypsin (Invitrogen). After an incubation periodof further 14 hrs at 37° C. IV titers and intracellular bacteria weredetermined as described in (Hrincius et al., 2010, Tuchscherr et al.,2011). IV titers are depicted as plaque forming units (pfu)/ml (A, C)and S. aureus titers are depicted as colonie forming units (CFU)/ml (B,D). Data represent the means±SD of two independent experiments (virustiter) and three independent experiments (bacterial titer) with twobiological samples. Statistical significance was evaluated by atwo-tailed two sample t-test (*p<0.05; **p<0.01; ***p<0.001).

FIG. 6: The viral neuraminidase inhibitor tamiflu reduces IV replicationbut enhances S. aureus load. Human lung epithelial cells were seeded in6-well plates (8×10⁵ cells/well) in 2 ml DMEM [10% FCS]. 16-20 hrs afterseeding, cells were rinsed and incubated with PBS/BA [0.2% bovine serumalbumin (BSA), 1 mM MgCl₂, 0.9 mM CaCl₂, 100 U/ml penicillin, 0.1 mg/mlstreptomycin] (500 μl per 6 well) or PBS/BA containing the Influenzavirus A/FPV/Bratislava/79 at a multiplicity of infection (MOI=0.001) at37° C. After 30 min incubation, the virus dilution was aspirated, cellswere rinsed with PBS and supplemented with invasion medium DMEM/INV [1%human serum albumin, 25 nmol/l HEPES] (2 ml per 6 well) with or withoutS. aureus 6850 (MOI=0.5) in presence of 2 μM tamiflu or Hepes (solventcontrol). 3 hrs post bacterial infection cells were treated withantibiotics to remove extracellular bacteria. Therefore cells wererinsed with PBS and subsequently incubated with DMEM/INVantibiotics (2μg/ml lysostaphin (Sigma)] (1 ml per 6 well) for 20 min at 37° C. Afteran additional wash with PBS cells were supplemented with DMEM/INVcontaining 2 μM tamiflu or Hepes. After an incubation period of further14 hrs at 37° C. IV titers and intracellular bacteria were determined asdescribed in (Hrincius et al., 2010; Tuchscherr et al., 2011).

FIG. 7: Titers of intracellular S. aureus 6850 are reduced upon LG-ASAtreatment. Human lung epithelial cells (A549) were infected with 0.5 MOIS. aureus 6850 DMEM/INV [1% human serum albumin, 25 nmol/l HEPES] for 3h in presence (A, C) and absence (B, D) of the indicated amounts ofLG-ASA. Three hours post infection an antibiotic wash was included usingDMEM/INVantibiotics [2 μg/ml lysostaphin (Sigma)] to removenon-internalized bacteria and subsequently cells were supplemented withDMEM/INV containing the indicated amounts of LG-ASA. Cell morphology wasmonitored by light microscopy (A, B) and amounts of internalizedbacteria were determined by serial dilution on agar plates 18 hours postinfection (C, D).

FIG. 8: Table 2: p38 inhibitors.

FIG. 9: Table 3: NFκB inhibitors.

FIG. 10: Table 4: NFκB inhibitors.

FIG. 11: Table 5: antibiotics.

FIG. 12: Time scale of the co-infection procedure in vitro. Human lungepithelial cells (A549) were infected with influenza A virus (IAV) for30 min at a multiplicity of infection (MOI) indicated, dissolved inPBS/BA [0.2% bovine serum albumin, 1 mM MgCl₂, 0.9 mM CaCl₂, 100 U/mlpenicillin, 0.1 mg/ml streptomycin] at 37° C. After 30 min incubation,the virus dilution was aspirated, cells were rinsed with PBS. Afterwardsbacterial infection with Staphylococcus aureus 6850 (S. aureus) wasperformed or cells were mock-treated. Therefore cells were supplementedwith invasion medium DMEM/INV [1% human serum albumin, 25 nmol/l HEPES]with or without S. aureus in addition to the indicated amounts ofinhibitor (U0126 or CI-1040) or solvent control. 3 hrs post bacterialinfection an antibiotic wash [DMEM, 10% FBS, 2 μg/ml Lysostaphin or 100μg/ml Gentamicin, 20 min] was introduced to remove non-internalizedbacteria. After an additional PBS wash, cells were supplemented withinfection medium DMEM (0.2% BA, 1 mM MgCl₂, 0.9 mM CaCl₂, 100 U/mlpenicillin, 0.1 mg/ml streptomycin) in presence or absence of theinhibitor and were incubated up to 18 hrs post viral infection at 37° C.

FIG. 13: Inhibition of the MEK/ERK signaling results in enhanced cellsurvival after singular and co-infection.

Human lung epithelial cells (A549) were infected with the avianinfluenza virus strain A/FPV/Bratislava/79 (H7N7) (FPV) or the humaninfluenza virus strain A/Wisconsin/67/2005 (H3N2) at a multiplicity ofinfection (MOI=0.01) (H7N7) or (MOI=0.5) (H3N2) at 37° C.. After 30 minthe virus dilution was removed, cells were rinsed with PBS andsupplemented with invasion medium DMEM/INV (containing 1% human serumalbumin, 25 nM HEPES) with or without S. aureus 6850 (MOI=0.1) inpresence of 50 μM U0126 or solvent control. 3 hrs post bacterialinfection cells were treated with DMEM/FBS containing 10% FBS, 2 μg/mllysostaphin for 20 min to remove non-internalized bacteria. After anadditional wash with PBS cells were supplemented with infection mediumDMEM/BA (0.2% BA, 1 mM MgCl₂, 0.9 mM CaCl₂, 100 U/ml penicillin, 0.1mg/ml streptomycin) containing 50 μM U0126 or solvent. After anincubation period of 18 hrs at 37° C. cell morphology was monitored bylight microscopy,

FIG. 14: Inhibition of MEK/ERK signaling results in reduced viral titersduring singular viral and co-infection.

Human lung epithelial cells (A549) were infected with the avianinfluenza virus strain A/FPV/Bratislava/79 (H7N7) (A, C) or the humaninfluenza virus strain A/Wisconsin/67/2005 (H3N2) (B, D) at amultiplicity of infection (MOI=0.01) (A, C), (MOI=0.5) (B, D) at 37° C.After 30 min the virus dilution was removed, cells were rinsed with PBSand supplemented with invasion medium DMEM/INV (containing 1% humanserum albumin, 25 nM HEPES) with or without S. aureus 6850 (MOI=0.1) inpresence of 50 μM U0126 or solvent control. 3 hrs post bacterialinfection cells were treated with DMEM/FBS containing 10% FBS, 2 μg/mllysostaphin for 20 min to remove extracellular bacteria. After anadditional wash with PBS cells were supplemented with infection mediumDMEM/BA (0.2% BA, 1 mM MgCl₂, 0.9 mM CaCl₂, 100U/ml penicillin, 0.1mg/ml streptomycin) containing 50 μM U0126 or solvent. After anincubation period of 18 hrs at 37° C. viral titers were determined bystandard plaque assay. Viral titers are depicted as plaque formingunits/ml (PFU/ml) with a linear (A, B) or logarithmic scale (C, D). Datarepresent the means±SD of four independent experiments with twobiological samples. Statistical significance was evaluated by atwo-tailed one sample t-test (*p<0.05; **p<0.01).

FIG. 15: MEK inhibition by administration of U0126 results in reducedbacterial growth.

Human lung epithelial cells (A549) were infected with the avianinfluenza virus strain A/FPV/Bratislava/79 (H7N7) or the human influenzavirus strain A/Wisconsin/67/2005 (H3N2) (A, C) at a multiplicity ofinfection (MOI=0.01) (H7N7) or (MOI=0.5) (H3N2) at 37° C. After 30 minthe virus dilution was removed, cells were rinsed with PBS andsupplemented with invasion medium DMEM/INV (containing 1% human serumalbumin, 25 nM HEPES) with or without S. aureus 6850 (MOI=0.1) (A) inpresence of 50 μM U0126 or solvent control. 3 hrs post bacterialinfection cells were treated with DMEM/FBS containing 10% FBS, 2 μg/mllysostaphin for 20 min (A, C) to remove non-internalized bacteria. Afteran additional wash with PBS cells were supplemented with infectionmedium DMEM/BA (0.2% BA, 1 mM MgCl₂, 0.9 mM CaCl₂, 100 U/ml penicillin,0.1 mg/ml streptomycin) containing 50 μM U0126 or solvent. Amounts ofinternalized bacteria were determined by serial dilution of cell lysateson agar plates 18 hrs post infection (A, C). The impact of U0126 onbacterial growth was analyzed by administration of U0126 as indicated toan over-night culture of S. aureus 6850 (100 CFU/ml). After 16 hrsserial dilutions were plated on BHI agar (B, D). Bacterial titers aredepicted as colony forming units/ml (CFU/ml) with a linear (A, B) orlogarithmic scale (C, D). Data represent the means±SD of four (A, C) orthree (B, D) independent experiments with two biological samples.Statistical significance was evaluated by a two-tailed two sample t-test(***p<0.001).

FIG. 16: Inhibition of the MEK/ERK signaling leads to reduction of viralproteins and pro-inflammatory chemokines.

Human lung epithelial cells (A549) were infected with the humaninfluenza virus strain A/Wisconsin/67/2005 (H3N2) at a multiplicity ofinfection (MOI=5) at 37° C. After 30 min the virus dilution was removed,cells were rinsed with PBS and supplemented with invasion mediumDMEM/INV (containing 1% human serum albumin, 25 nM HEPES) with orwithout S. aureus 6850 (MOI=50) in presence of 50 μM U0126 or solventcontrol. 3 hrs post bacterial infection cells were treated with DMEM/FBScontaining 10% FBS, 100 μg/ml Gentamicin for 30 min to removeextracellular bacteria. After an additional wash with PBS cells weresupplemented with infection medium DMEM/BA (0.2% BA, 1 mM MgCl₂, 0.9 mMCaCl₂, 100 U/ml penicillin, 0.1 mg/ml streptomycin) containing 50 μMU0126 or solvent. After an incubation period of 8 hrs at 37° C. mRNAlevels of CCL3 and CCL5 were analyzed by qRT-PCR with specific primers(A, B). Viral protein expression (PB-1) and the level of ERK-1/2phosphorylation, as well as bacterial cell wall components (PGN) weredetermined by western blot analysis (C). Data represent preliminaryresults of three biological samples and two technical replicates withinone experiment.

FIG. 17: Administration of U0126 leads to reduced bacterial titers invivo independent of viral titers.

12 weeks old Balb/C mice were infected with 50 PFU of the influenzavirus strain A/Puerto Rico/8/34 (PR8, H1N1) on day 0 (anesthesized withIsoflurane). Starting on day 1 mice were treated daily with i.p.injection of 30 mg/kg/day U0126 or solvent control (10% DMSO, 30%Cremophor EL, 60% PBS). On day 3 mice were infected with 5*10⁷ CFU ofStaphylococcus aureus 6850 under anesthesia with Isoflurane and directlytreated with U0126 or solvent control. On day 4 lungs were removed andhomogenized in PBS (0.1 g per 1000 μl PBS). For calculation of bacterialtiters serial dilutions of the homogenate were plated on BHI agar. Fordetermination of viral titers a standard plaque assay was performed.Statistical analysis was done using Mann-Whitney U Test (*p<0.05).

FIG. 18: The specific MEK inhibitor CI-4040 reduces viral titers insingular and co-infection.

Human lung epithelial cells (A549) were infected with the avianinfluenza virus strain A/FPV/Bratislava/79 (H7N7) (A, C) or the humaninfluenza virus strain A/Puerto Rico/8/34 (H1N1) (B, D) at amultiplicity of infection (MOI=0.0) at 37° C. After 30 min the virusdilution was removed, cells were rinsed with PBS and supplemented withinvasion medium DMEM/INV (containing 1% human serum albumin, 25 nMHEPES) with or without S. aureus 6850 (MOI=0.1) in presence of 10 μMCI-1040 or solvent control. 3 hrs pod bacterial infection relic weretreated with DMEM/FBS containing 10% FBS, 2 μg/ml lysostaphin for 20 minto remove extracellular bacteria. After an additional wash with PBScells were supplemented with infection medium DMEM/BA (0.2% BA, 1 mMMgCl₂, 0.9 mM CaCl₂, 100 U/ml penicillin, 0.1 mg/ml streptomycin)containing 10 μM CI-1040 or solvent. After an incubation period of 18hrs at 37° C. viral titers were determined by standard plaque assay.Viral titers are depicted as plaque forming units/ml (PFU/ml) with alinear (A, B) or logarithmic scale (C, D). Data represent the means oftwo independent experiments with two biological samples.

FIG. 19: Administration of CI-1040 has an inhibitory effect on bacterialgrowth in vitro.

The impact of CI-1040 on bacterial growth was analyzed by administrationof CI-1040 in different concentrations (as indicated) to an over-nightculture of S. aureus 6850 (100 CFU/ml). After 16 hrs serial dilutionswere plated on BHI agar (A, B). Bacterial titers are depicted as colonyforming units/ml (CFU/ml) with a linear (A) or logarithmic scale (B).Data represent preliminary data with two biological samples.

FIG. 20: Treatment with the specific MEK inhibitor Cobimetinib(GDC-0973) reduces pathogen load in vivo.

8 weeks old Balb/C mice (5 per group) were infected with 50 PFU ofinfluenza virus strain A/Puerto Rico/8/34 (PR8, H1N1) on day 0(anesthesized with Isoflurane). 6 hrs post viral infection mice weretreated with oral administration of 10 mg/kg/day Cobimetinib or solventcontrol (10% DMSO, 5% Tween 20, 85% PBS), Treatment was then performeddaily. On day 3 mice were infected with 5*10⁷ CFU of Staphylococcusaureus 6850 under anesthesia with Isoflurane and 6 hrs later treatedwith Cobimetinib or solvent control. On day 4 lungs were removed andhomogenized in PBS (0.1 g per 1000 μl PBS). For calculation of bacterialtiters serial dilutions of the homogenate were plated on BHI agar. Fordetermination of viral titers a standard plaque assay was performed.Statistical analysis was done using Mann-Whitney U test.

FIG. 21: LG-ASA improves cell morphology upon infection with influenza Avirus (IAV) and/or Staphylococcus aureus (S. aureus)

Human lung epithelial cells (A549) were infected with the influenzavirus strain A/Puerto Rico/8/34 (H1N1) at a multiplicity of infection(MOI=0.1) dissolved in PBS/BA [0.2% bovine serum albumin, 1 mM MgCl₂,0.9 mM CaCl₂, 100 U/ml penicillin, 0.1 mg/ml streptomycin] at 37° C.After 30 min incubation, the virus dilution was aspirated, cells wererinsed with PBS and supplemented with invasion medium DMEM/INV [1% humanserum albumin, 25 nmol/l HEPES] with or without S. aureus SH1000(MOI=0.1) in presence of 5 mM LG-ASA or solvent control. 3 hrs postbacterial infection cells were treated with antibiotics [DMEM, 10% FBS,2 μg/ml lysostaphin, 20 min] to remove extracellular bacteria. After anadditional wash with PBS cells were supplemented with DMEM/INVcontaining 5 mM LG-ASA or solvent. After an incubation period of 18 hrsat 37° C. cell morphology was monitored by light microscopy.

FIG. 22: The NFκB inhibitor LG-ASA reduces influenza virus titers and S.aureus load

Human lung epithelial cells (A549) were infected with the influenzavirus strain A/Puerto Rico/8/34 (H1N1) at a multiplicity of infection(MOI=0.1) dissolved in PBS/BA [0.2% bovine serum albumin, 1 mM MgCl₂,0.9 mM CaCl₂, 100 U/ml penicillin, 0.1 mg/ml streptomycin] at 37° C.(A-H). After 30 min incubation, the virus dilution was aspirated, cellswere rinsed with PBS and supplemented with invasion medium DMEM/INV [1%human serum albumin, 25 nmol/l HEPES] with or without S. aureus SH1000(MOI=50) (A, B), (MOI=0.01) (C-D), S. aureus 6850 (MOI=50) (E, F),(MOI=0.1) (G, H) in presence of 5 mM LG-ASA or solvent control. 3 hrspost bacterial infection cells were treated with antibiotics [DMEM, 10%FBS, 2 μg/ml lysostaphin, 20 min] to remove extracellular bacteria.After an additional wash with PBS cells were supplemented with DMEM/INVcontaining 5 mM LG-ASA or solvent. After an incubation period of 8 hrs(A, B, E, F) or 18 hrs (C, D, G, H) at 37° C. IAV titers (A, C, E, G)were determined by standard plaque assays. Cells were lysed by ahypotonic shock and amounts of internalized bacteria (B, D, F, H) weredetermined by serial dilution on agar plates. Data represent themeans±SD of three (A-H) with two biological samples. Statisticalsignificance was evaluated by a two-tailed one sample t-test (*p<0.05;**p<0.01).

FIG. 23: The NFκB inhibitor LG-ASA reduces influenza virus titers and S.aureus load

This figure presents data obtained as in FIG. 22 in a different way. Inparticular, the untreated controls of each experiment were arbitrarilyset as 100% and then the means were calculated. Statistical significancewas evaluated by a two-tailed one sample t-test (*p<0.05; ** p<0.01).

FIG. 24: Inhibition of NFκB signaling results in reduced bacterialinternalisation

Human lung epithelial cells (A549) were preincubated with 5 mM (A-D) and10 mM (C, D) LG-ASA for 4 h and then infected with S. aureus 6850(MOI=50) (A, B) for 30-120 min and USA 300 (MOI=5) (C, D) for 120 min inpresence and absence of the indicated amounts of LG-ASA dissolved inDMEM/INV [1% human serum albumin, 25 nmol/l HEPES]. 30-120 min postinfection an antibiotic wash [DMEM, 10% FBS, 2 μg/ml lysostaphin, 20min] was included to remove non-internalized bacteria. Cells were washedwith PBS three times and lysed by hypotonic shock. Amounts ofinternalized bacteria were determined by serial dilution on agar plates(A-D). Data (A, C) represent the means±SD of three independentexperiments with two biological samples. In FIG. 24 B, D the untreatedcontrols of each experiment were arbitrarily set as 100% and then themeans were calculated. Statistical significance was evaluated by atwo-tailed one sample t-test (*p<0.05; **p<0.01; ***p<0.001).

FIG. 25: Stimulation of NFκB signaling results in enhanced bacterialinternalization Human lung epithelial cells (A549) were preincubatedwith 5 mM LG-ASA and 2.5 ng/ml TNF-alpha for 4 h and then infected with(A) S. aureus 6850 or (B) S. aureus USA300 (MOI=5) for 120 min inpresence and absence of the indicated amounts of LG-ASA and TNF-alphadissolved in DMEM/INV [1% human serum albumin, 25 nmol/l HEPES] (A). 120min post infection an antibiotic wash [DMEM, 10% FBS, 2 μg/mllysostaphin, 20 min] was included to remove non-internalized bacteria.Cells were washed with PBS three times and lysed by hypotonic shock.Amounts of internalized bacteria were determined by serial dilution onagar plates (A) 120 min post infection. (A) Data represent the means±SDof four independent experiments with two biological samples. Statisticalsignificance was evaluated by an one-way ANOVA followed by a multiplecomparison test (*p<0.05; **p<0.01; ***p<0.001). (B) Data represent themeans±SD of three independent experiments with three biological sampleswhereby the untreated controls of each experiment were arbitrarily setas 100% and then the means were calculated.

FIG. 26: Treatment of IAV/S. aureus co-infected mice with LG-ASA resultsin enhanced survival and reduced body weight loss

(A) BALB/c mice (4 mice per group) were infected with 50 PFU of theinfluenza virus A/Puerto Rico/8/34 at day 0. On day 6 after influenzavirus infection mice were additionally infected with 10⁸ CFU S. aureus6850. On day 7 after influenza virus infection co-infected mice weretreated once a day with LG-ASA (1M, 10 min) via inhalation. Survival wasmonitored for 14 days. While (4/4) (black line) untreated co-infectedmice died 1 day after co-infection, (2/4) LG-ASA treated co-infectedmice survived (grey line).(B) Two independent experiments are depicted. 9 weeks old Balb/C mice (4mice per group) were infected with 50 PFU of influenza virus A/PuertoRico/8/34 on day 0 (anesthesized with Isoflurane) in the morning. 6 hrspost viral infection mice were weighed and treated with aerosolic H₂O or1M LG-ASA in an inhalation chamber for 10 min. This treatment was alsoperformed on day 1, 2 and 3 at the same time as on day 0. On day 3 inthe morning mice were infected with 5*10⁷ CFU of Staphylococcus aureus6850 under anesthesia with Isoflurane. On day 4 mice were weighed thelast time. Statistical analysis was done using Mann-Whitney U Test(*p<0.05).

DETAILED DESCRIPTION OF THE INVENTION/DETAILED DESCRIPTION OF APREFERENTIAL EMBODIMENT

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

The above being said, the present invention relates to a MEK inhibitor,p38 inhibitor and/or NFκB inhibitor for use in a method for theprophylaxis and/or treatment of a co-infection comprising a bacterialinfection and an influenza virus infection.

In addition the present invention relates to a MEK inhibitor, p38inhibitor and/or NFκB inhibitor for use in a method for the prophylaxisand/or treatment of a bacterial infection.

When used herein, a “MEK inhibitor” may also be designated as a MitogenActivated Protein kinase (MAPK) kinase inhibitor. It is known that in aMAPK pathway, a MAPK kinase kinase (MAPKKK) activates a MAPK kinase(MAPKK) which in turn activates a MAPK which transduces a signal to, forexample, a transcription factor or other kinases or effector/signaltransducing protein; see, for example, FIG. 1 of Fremin and Meloche(Fremin and Meloche (2010), J. Hematol. Oncol. 11;3:8). MEK inhibitorsof the invention preferably inhibit MEK1/2 of a subject, such as amammal or bird as described herein. However, it may be that a MEKinhibitor of the invention does not only inhibit a MEK, preferablyMEK1/2, but also its upstream kinase (i.e. MAPKKK), thereby exerting adual inhibition. Without being bound by theory, PLX-4032 may be such adual inhibitor. Hence, a MEK inhibitor of the invention may in apreferred aspect by a dual inhibitor, thereby inhibiting a MEK,preferably MEK1/2 and the corresponding upstream MAPKKK. MEK1/2 is theMAPKK in the Ras/Raf pathway, whereby Ras/Raf acts as MAPKKK and ERK1/2acts as MAPK.

A MEK inhibitor can be a small molecule, large molecule, peptide,oligonucleotide, and the like. The MEK inhibitor may be a protein orfragment thereof or a nucleic acid molecule. Also included by the termMEK inhibitor is a pharmaceutically acceptable salt of the MEKinhibitor.

The determination of whether or not a compound is a MEK inhibitor iswithin the skill of one of ordinary skill in the art. In one embodiment,the MEK inhibitors are selected from the group consisting of thecompounds/inhibitors listed in table 1.

The MEK inhibitors of the invention are selected preferably from U0126,PLX-4032, AZD6244, AZD8330, AS-703026, GSK-1120212, RDEA-119,RO-5126766, RO-4987655, CI-1040, PD-0325901, GDC-0973, TAK-733, PD98059,PD184352 ARRY-438162 and PF-3644022, preferably AZD8330, GSK-1120212,U0126, GDC-0973, CI-1040, PD0325901, ARRY-438162, PF-3644022 and AZD6244and most preferably U0126, CI-1040, GDC-0973 (Cobimetinib), AZD8330,GSK-1120212, most preferably U0126, GDC-0973, CI-1040, AZD8330 andGSK-1120212.

Some of these inhibitors are further described in table 1 below.

TABLE 1 MEK inhibitors.

Structural formula I Cl-1040 2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)- 3,4-difluorobenzamide

Structural formula II PD0325901 (R)-N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)benzamide

Structural formula III AZD6244 6-(4-bromo-2-chlorophenylamino)-7-fluoro-N-(2-hydroxyethoxy)-3-methyl-3H-benzo[d]imidazole-5-carboxamide

Structural formula IV GDC-0973 [3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl] [3-hydroxy-3-[(2S)-2-piperidinyl]-1-azetidinyl]methanone

Structural formula V RDEA-119 (S)-N-(3,4-difluoro-2-(2-fluoro-4-iodophenyl- amino)-6-methoxyphenyl)-1-(2,3-dihydroxypropyl)cyclopropane- 1-sulfonamide

Structural formula VI GSK-1120212 N-(3-(3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)- 6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido [4,3-d]pyrimidin-1(2H)- yl)phenyl)acetamide

Structural formula VII AZD8330 2-(2-fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo- 1,6-dihydropyridine-3-carboxamide

Structural formula VIII RO5126766 C20H16FN5O5S

Structural formula IX RO4987655 C20H19F3IN3O5

Structural formula X TAK-733 (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8- methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione

Structural formula XI PLX-4032 N-[3-[[5-(4-Chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3- yl]carbonyl]-2,4-difluorophenyl]-1-propanesulfonamide Zelboraf (vemurafenib)

Structural formula XII AS703026 (S)-N-(2,3-dihydroxypropyl)-3-(2-fluoro-4-iodophenylamino) isonicotinamide

Structural formula XIII PD98059 2-(2-amino-3-methoxyphenyl)-4H-chromen-4-one

Structural formula XIV PD184352 2-(2-chloro-4-iodophenylamino)-N-(cyclopropylmethoxy)- 3,4-difluorobenzamide

Also preferred is a selection from PLX-4032, AZD6244, AZD8330, GDC-0973,RDEA119, GSK1120212, RO51267766, RO4987655, TAK-733, and AS703026. Evenmore preferably, they are selected from AZD6244, AZD8330, GSK1120212 andPLX-4032 or from PD-0325901, AZD-6244, AZD-8330 and RDEA-119. These MEKinhibitors are known in the art and, for example, described in Table 1of Fremin and Meloche (2010), J. Hematol. Oncol. 11;3:8.

More information on some of these inhibitors can also be obtained fromArthur and Ley (2013) Mitogen-activated protein kinases in innateimmunity; Nature Reviews Immunology 13,679-692 (2013).

Indeed, as demonstrated in the appended Examples, the MEK inhibitorU0126 and CI-1040 disclosed herein show an effect in co-infectionscenarios as well as on bacterial infection alone.

Also a p38 inhibitor is provided for use in the methods for theprophylaxis and/or treatment of a co-infection or bacterial infection ofthe present invention. A “p38 MAP kinase inhibitor” is well known in theart. The terms “p38 inhibitor,” “p38 kinase inhibitor,” and “p38 MAPkinase inhibitor” are used interchangeably herein. In the context of thepresent invention a p38 MAP kinase inhibitor inhibits p38 MAP kinase.Preferably, the p38 MAP kinase inhibitor inhibits one of the isoforms ofp38 MAP kinase, preferably one of the four isoforms (α, β, γ or δ) ofp38 MAP kinase with the α-isoform being preferred, more preferably itinhibits any combination of two isoforms of p38 MAP kinase, even morepreferably it inhibits any combination of three isoforms of p38 MAPkinase and most preferably, it inhibits all isoforms or the α, β, γ andδ isoform of p38 MAP kinase. In some embodiments, the p38 MAP kinaseinhibitor inhibits the isoform of p38 that is involved in inflammatorydiseases, autoimmune diseases, destructive bone disorders, proliferativedisorders, infectious diseases, viral diseases or neurodegenerativediseases. It is reported that the α-isoform of p38 MAP kinase isinvolved in inflammation, proliferation, differentiation and apoptosis,whereas the biological functions of p38β, p38δ and p38γ are not yetunderstood completely. Accordingly, it is preferred herein that the p38MAP kinase inhibitor inhibits the α-isoform.

A p38 MAP kinase inhibitor can be a small molecule, large molecule,peptide, oligonucleotide, and the like. The p38 MAP kinase inhibitor maybe a protein or fragment thereof or a nucleic acid molecule. Alsoincluded by the term p38 inhibitor is a pharmaceutically acceptable saltof the 38 inhibitor.

The determination of whether or not a compound is a p38 kinase inhibitoris within the skill of one of ordinary skill in the art.

There are many examples of p38 inhibitors in the art. U.S. Pat. Nos.5,965,583, 6,040,320, 6,147,096, 6,214,830, 6,469,174, 6,521,655disclose compounds that are p38 inhibitors. U.S. Pat. Nos. 6,410,540,6,476,031 and 6,448,257 also disclose compounds that are p38 inhibitors.Similarly, U.S. Pat. Nos. 6,410,540, 6,479,507 and 6,509,361 disclosecompounds that are asserted to be p38 inhibitors. U.S. PublishedApplication Nos. 20020198214 and 20020132843 disclose compounds that aresaid to be p38 inhibitors. Another p38 MAP kinase inhibitor is BIRB 796BS(1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)-naphthalen-1-yl]-urea); see Branger (2002), J. Immunol. 168:4070-4077 orU.S. Pat. No. 6,319,921 for further p39 MAP kinase inhibitors.

Other p38 MAP kinase inhibitors are AMG 548 (Amgen), BIRB 796(Boehringer Ingelheim), VX 702 (Vertex/Kissei), SCIO 469, SCIO 323(Scios Inc.), SB 681323 (GlaxoSmithKline), PH-797804 (Pfizer) andOrg-48762-0 (Organon NV); see, for example, Lee and Dominguez in CurrMed Chem. 2005; 12(25):2979-2994 and Dominguez in Curr Opin Drug DiscovDevel. 2005 July; 8(4):421-430.

According to the present invention, the inhibitor may exhibit itsregulatory effect upstream or downstream of p38 MAP kinase or on p38 MAPkinase directly, with the latter mode of action being preferred.Examples of inhibitor regulated p38 MAP kinase activity include thosewhere the inhibitor may decrease transcription and/or translation of p38MAP kinase, may decrease or inhibit post-translational modificationand/or cellular trafficking of p38 MAP kinase, or may shorten thehalf-life of p38 MAP kinase. The inhibitor may also reversibly orirreversibly bind p38 MAP kinase, inhibit its activation, inactivate itsenzymatic activity, or otherwise interfere with its interaction withdownstream substrates.

The four isoforms of the p38 MAP kinase share a high level of sequencehomology. The alpha and beta isoforms of the p38 MAP kinase are closelyrelated while the gamma and delta isoforms are more divergent. Given thehigh degree of structural similarity, it is not surprising that certaincompounds with the ability to inhibit one p38 MAP kinase isoform canoften inhibit other isoforms of the MAP kinase. Accordingly, in someembodiments, an inhibitor of p38 MAP kinase that is specific for theα-isoform of the kinase possesses at least three categories ofstructural features that are theorized to permit isoform specificinhibition.

Selective binding of a candidate p38 MAP kinase inhibitor can bedetermined by a variety of methods. The genes for the various isoformsof p38 MAP kinase are known in the art. One of ordinary skill in the artcould readily clone and express the various isoforms of the kinase,purify them, and then perform binding studies with candidate compoundsto determine isoform binding characteristics. This series of experimentswas performed for the α-isoform of p38 MAP kinase and provided in U.S.Pat. No. 6,617,324 B1.

Another kinase selectivity assay is described in Mihara (2008), Br. J.Pharmacol. 154(1):153-164. In some embodiments herein, a p38 MAP kinaseinhibitor inhibits one of the four isoforms of p38 MAP kinase, morepreferably it inhibits any combination of two isoforms of p38 MAPkinase, even more preferably it inhibits any combination of threeisoforms of p38 MAP kinase, e.g., p38-α(MAPK14), -β(MAPK11), -γ(MAPK12or ERK6). Alternatively, but also preferred, it inhibits all fourisoforms of p38 MAP kinase.

In one embodiment, the p38 inhibitor is selected from the groupconsisting of the inhibitors listed in table 2 (FIG. 8). In anotherembodiment, the p38 inhibitor is selected from the group consisting ofSB202190, LY2228820, CAY10571, SB 203580, Tie2 Kinase Inhibitor,2-(4-Chlorophenyl)-4-(fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one,CGH 2466, SB220025, Antibiotic LL Z1640-2, TAK 715, SB202190hydrochloride, SKF 86002, AMG548, CMPD-1, EO 1428, JX 401, ML 3403, RWJ67657, SB 202190, SB 203580, SB 203580 hydrochloride, SB 239063, SCIO469, SX 011, TAK 715, Pamapimod, Losmapimod (GW856553), Dilmapimod(SB681323), VX 702, VX 745, Doramapimod (BIRB 796), BMS-582949,ARRY-797, PH797804, SCIO-469, preferably VX-702, SB202190, Pamapimod,Losmapimod (GW856553), Dilmapimod (SB681323), Doramapimod (BIRB 796),BMS-582949, ARRY-797, PH797804 and SCIO-469.

More information on some of these inhibitors can also be obtained fromArthur and Ley (2013) Mitogen-activated protein kinases in innateimmunity; Nature Reviews Immunology 13,679-692 (2013).

In addition to the MEK inhibitor and the p38 inhibitor, the presentinvention is also directed to a NFκB (NFκB/NFkappaB) inhibitor for usein the methods for the prophylaxis and/or treatment of a co-infection orbacterial infection of the present invention. The determination ofwhether or not a compound is a NFκB inhibitor is within the skill of oneof ordinary skill in the art.

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells)is a protein complex that controls transcription of DNA. NF-κB is foundin almost all animal cell types and is involved in cellular responses tostimuli such as stress, cytokines, free radicals, ultravioletirradiation, oxidized LDL, and bacterial or viral antigens. VertebrateNFκB transcription complexes can be any of a variety of homo- andheterodimers formed by the subunits p50 (NFκB1), p52 (NFκB2), c-Rel,RelA (p65) and RelB (Gilmore T D. (2006) Oncogene 25: 6680-6684). Thesecomplexes bind to DNA regulatory sites called κB sites, generally toactivate specific target gene expression. In most cell types, NF-κBdimers are located in the cytoplasm in an inactive form throughassociation with any of several IκB inhibitor proteins (IκB α, β, ϵ, γ,p105 and p100). In response to a wide array of stimuli IκB is rapidlyphosphorylated, ubiquitinated and degraded by the proteasome. The freedNF-κB dimer then translocates to the nucleus where it can modulatespecific gene expression.

The phosphorylation and degradation of IκB is important for theregulation of NFκB complexes, which is mediated by the IκB kinase (IKK)complex containing two kinase subunits, IKKα and IKKβ, and an associatedscaffold-like regulatory protein called NEMO (aka IKKγ) (Gilmore andHerscovich (2006) Inhibitors of NF-κB signaling: 785 and countingOncogene. 25, 6887-6899). Notably, as for example shown in Example 3 ofthe present invention, NE-κB signaling is also important for bacterial(such as S. aureus) internalisation into cells.

According to the present invention, the inhibitor may exhibit itsregulatory effect upstream or downstream of NFκB or directly on NFκB,with the latter mode of action being preferred. Examples of inhibitorsregulating NFκB activity include those where the inhibitor may decreasetranscription and/or translation of NFκB, or may shorten the half-lifeof NFκB. The inhibitor may also reversibly or irreversibly bind NFκB,inhibit its activation, inactivate its activity, or otherwise interferewith its interaction with downstream targets, such as targets on genes.Also, an NFκB inhibitor can inhibit protein kinases such as moleculeswhich inhibit IkBa phosphorylation by e.g. IKK inhibition. Compoundsthat have such an activity are SC-893, BMS-345541, which may serve asreference compounds. Also a NFκB inhibitor may inhibit proteinphosphatases, or inhibit the proteasome, or ubiquitination. Examples ofsuch NFκB inhibitors, which may also serve as reference compoundsinclude protein tyrosine phosphatase inhibitors, boronate, bortezomib,NPI-0052. Alternatively, a NFκB inhibitor may block the nucleartranslocation of NFκB, or its binding to DNA. Examples of suchinhibitors include, which may also serve as reference compounds, SN50,dehydroxymethylepoxyquinomicin and NFκB decoy ODNs. Further informationon inhibitor of NFκB can be obtained from Gupta et al. (2010) (Gupta etal. (2010) Inhibiting NFκB activation by small molecules as atherapeutic strategy. Biochim Biophys Acta. 1799(10-12): 775-787).

A NFκB inhibitor can be a small molecule, large molecule, peptide,oligonucleotide, and the like. The NFκB inhibitor may be a protein orfragment thereof or a nucleic acid molecule. Also included by the termNFκB inhibitor is a pharmaceutically acceptable salt of the NFκBinhibitor. In one embodiment, the NFκB inhibitor is selected from thegroup consisting of the inhibitors/molecules as listed in tables 3 and 4in FIGS. 9 and 10. In another embodiment, the NFκB inhibitor is selectedfrom the group consisting of the inhibitors/molecules as listed in table3 in FIG. 9. In another embodiment, the NFκB inhibitor is selected fromthe group consisting of the inhibitors/molecules as listed in table 4 inFIG. 10.

In another embodiment, the NFκB inhibitor is selected from the groupconsisting of LASAG, SC75741 (and derivatives), MG 132, TPCA-1, PCTC,IMD 0354, Luteolin, Caffeic acid phenethyl ester, Cardamonin, PF 184,IKK 16, SC 514, Withaferin A, Arctigenin, Bay 11-7085, PSI, PR 39, Ro106-9920, Bay 11-7821, ML-130, Celastrol, Tanshinone IIA, HU 211,Gliotoxin, CID 2858522, Honokiol, Andrographolide, 10Z-Hymenialdisine,ACHP, Pristimerin, Sulfasalazine, ML 120B dihydrochioride, Amlexanox,9-Methylstreptimidone, N-Stearoyl phytosphingosine,2-(1,8-naphthyridin-2-yl)-Phenol, 5-Aminosalicylic acid, BAY 11-7085,Ethyl 3,4-Dihydroxycinnamate, Helenalin, NF-κB Activation Inhibitor II,JSH-23, Glucocorticoid Receptor Modulator, CpdA, PPM-18, aspirin (ASA),Pyrrolidinedithiocarbamic acid ammonium salt, (R)-MG132, SC75741 (andderivatives), Rocaglamide, Sodium salicylate, QNZ, PS-1145, CAY10512,bortezomib, salsalate, resveratrol, LASAG, deoxyspergualin, sulindac,thalidomide, AGRO-100, CHS 828 and/or Curcumin, preferably bortezomib,curcumin, salsalate, resveratrol, sodium salicylate, LASAG, ASA,deoxyspergualin, sulindac, thalidomide, AGRO-100, CHS 828 even morepreferably SC75741 (and derivatives) ASA and LASAG and most preferablyLASAG.

With the term “SC75741” or “SC75741 (and derivates)” in addition toSC75741 also derivates of SC75741 are envisaged by the presentinvention.

In general a person skilled in the art knows how to find out if acompound is an MEK inhibitor, p38 inhibitor and/or NFκB inhibitor. Afurther example of how one could determine if a compound is a MEKinhibitor and/or p38 inhibitor would be to isolate the MEK and/or p38NFκB protein. The protein can be isolated from cells where the MEKand/or p38 protein is naturally expressed or where it has beenoverexpressed by means of transfection of an oligonucleotide orinfection with a virus that directs the expression of the MEK and/or p38protein, Additionally, MEK and/or p38 protein can also be expressedrecombinantly. Upon isolating the protein a person of ordinary skill inthe art can measure the activity of the kinase in the presence orabsence of a potential MEK and/or p38 inhibitor. If the kinase activityis less in the presence than in the absence of an alleged inhibitor,that inhibitor is a MEK and/or p38, respectively.

If acting on MEK and/or p38 directly, the inhibitor should exhibit anIC50 value of about 5 μM or less, preferably 500 nm or less, morepreferably 100 nm or less. In a related embodiment, the inhibitor shouldexhibit an IC50 value relative to the p38-α isoform that is preferablyat least ten fold less than that observed when the same inhibitor istested against other p38 MAP kinase isoforms in the same or comparableassay. It should be noted that IC50 values are assay dependent and maychange from determination to determination. It is more important to lookat relative relationships of compounds' IC50 values rather than theexact values themselves.

An IC50 is the concentration of compound which inhibits the enzyme to50% of the activity as measured in the absence of an inhibitor.

IC50 values are calculated using the concentration of inhibitor thatcauses a 50% decrease as compared to a control. IC50 values are assaydependent and will vary from measurement to measurement. As such, IC50values are relative values. The values assigned to a particularinhibitor are to be compared generally rather than on an absolute basis.

Samples or assays comprising MAP and/or MAPK kinase that are treatedwith a potential activator, inhibitor, or modulator are compared tocontrol samples without the inhibitor, activator, or modulator toexamine the extent of inhibition. Control samples (untreated withinhibitors) can be assigned a relative MAP and/or MAPK kinase activityvalue of 100%. Inhibition of MAP and/or MAPK kinase is achieved when theMAP and/or MAPK kinase activity value relative to the control is about80%, optionally 50% or 25-0%. Activation of MAP and/or MAPK kinase isachieved when the MAP kinase activity value relative to the control is110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.Exemplary MAP kinase binding activity assays of the present inventionare: a MAP and/or MAPK kinase ligand blot assay (Aymerich et al., InvestOpthalmol Vis Sci. 42:3287-93, 2001); a MAP and/or MAPK kinase affinitycolumn chromatography assay (Alberdi et al., J Biol Chem. 274:31605-12,1999) and a MAP and/or MAPK kinase ligand binding assay (Alberdi et al.,J Biol Chem. 274:31605-12, 1999). Each incorporated by reference intheir entirety.

Also the selectivity of the inhibitors may be measured by a kinaseselectivity assay is described in Mihara (2008), Br. J. Pharmacol.154(1):153-164.

In the case of the NFκB inhibitor one can measure for example the geneproducts (proteins) of target genes of NFκB in a non-treated controlcell and compare the expression of these target gene products to a cell,which has been treated with a NFκB Inhibitor. Some target genes aredescribed in Oeckinghaus and Ghosh (2009) The NF-κB Family ofTranscription Factors and Its Regulation. Cold Spring Harb PerspectBiol. October 2009; 1(4): a000034.

The expression level is reduced, when the cell treated with theinhibitor. Other strategies may be to detect IkBa degradation togetherwith p-p65 accumulation and nuclear translocation of NFκB byWesternblot. Also NFκB interactions with DNA of cells not treated withan inhibitor compared to cells that have been treated with an inhibitormay be analysed by using an electrophoretic mobility shift assay (EMSA).

The inhibitory properties of a molecule can also be analysed bycomparing its action to a reference compound. A “reference compound” asreferred to herein means a compound, which may be used as a positivecontrol for the determination if a molecule has MEK inhibitor, p38inhibitor and/or NFκB inhibitor properties. As such also any of theinhibitors listed herein may be used as such a reference compound. Apossible test may be one in which cells, which are e.g. stimulated toactivate the MEK, p38 and or NFκB pathway are treated with a referencecompound and in parallel e.g. in a different well with a compound ofinterest.

The inhibitors of the present invention can be used in a method fortreating and/or prophylaxis. As such the term “treating” or “treatment”includes administration of a MEK inhibitor, p38 Inhibitor, and/or NFκBinhibitor preferably in the form of a medicament, to a subject sufferingfrom a coinfection comprising a bacterial infection and an influenzavirus infection for the purpose of ameliorating or improving symptoms.Similarly included is the administration of a MEK inhibitor, p38Inhibitor, and/or NFκB inhibitor preferably in the form of a medicament,to a subject suffering from a bacterial infection for the purpose ofameliorating or improving symptoms.

Furthermore, the terms “prophylaxis” as used herein, refers to anymedical or public health procedure whose purpose is to prevent adisease. As used herein, the terms “prevent”, “prevention” and“preventing” refer to the reduction in the risk of acquiring ordeveloping a given condition, namely a coninfection comprising aninfluenza virus infection and a bacterial infection or a bacterialinfection alone. Also meant by “prophylaxis” is the reduction orinhibition of the recurrence of a coninfection comprising an influenzavirus infection and a bacterial infection or a bacterial infection alonein a subject.

The inhibitors of the present invention are effective in treating acoinfection. A “co-infection” as used herein comprises an influenzavirus infection and a bacterial infection. Such a coinfection can takeplace by simultaneous infection of a host e.g. a subject and/or singlecell with a bacterium and an influenza virus. It can also be that a hoste.g. a subject and/or cell is simultaneously infected with one or moreviral particles and one or more bacteria. However, such a coinfectioncan also take place sequentially. In such a case is firstly infectedwith one or more viral particles and later in time the same host and/orcell becomes infected with one or more bacteria or vice versa. The timeperiod between the two infections can be a time period of at most 14days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6days, 5 days, 4 days, 3 days, 2 days, 1 day, 12 hours, 6 hours, 3 hours,1.5 hours or at minimum 30 minutes.

Such a situation may also be a superinfection, in which a secondinfection is superimposed on an earlier one especially by a differentmicrobial agent of exogenous or endogenous origin that is resistant tothe treatment used against the first infection.

Within the co-infection the influenza virus infection can be mediated byinfluenza A virus or influenza B virus, preferably the influenza A virusis H1N1, H2N2, H3N2, H6N1, H7N7, H7N9, H9N2 H10N7, H10N8 or H5N1. In oneembodiment, the influenza A virus is H1N1. In other embodiments, theinfluenza A virus is H3N2, H5N1 and H7N9. In additional embodiments, theinfluenza A virus is H3N2, H5N1, H1N1 and H7N9.

The present invention also relates to a “bacterial infection” which cantake place in the setting of a co-infection described above or can occuras the only infection present in a host e.g. a subject and/or cell. Thebacterial infection can be mediated by any bacterium, preferably it ismediated by a bacterium selected from the group consisting ofStaphylococcaceae, Streptococcaceae, Legionellaceae, Pseudomonadaceae,Chlamydiaceae, Mycoplasmataceae, Enterobacteriaceae, Pseudomonadalesand/or Pasteurellaceae.

In other embodiments the bacterial infection is mediated by a bacteriumselected from the group consisting of of Staphylococcus, preferablyStaphylococcus aureus, methicillin sensitive and methicillin resistantStaphylococcus aureus, Panton-Valentine leukocidin (PVL)-expressingStaphylococcus aureus and/or Streptococcaceae, preferably Streptococcusmitis, Streptococcus pyogenes or Streptococcus pneumonia, Legionella,preferably Legionella pneumophila, Pseudomonas, preferably Pseudomonasaeruginosa, Chlamydophila, preferably Chlamydophila pneumonia,Mycoplasma, preferably Mycoplasma pneumonia, Klebsiella, preferablyKlebsiella pneumonia, Moraxella, preferably Moraxella catarrhalis and/orHaemophilus, preferably Haemophilius influenza. Preferably the bacteriumis selected from the group consisting of Staphylococcus aureus,Streptococcus pneumonia or Haemophilius influenza. Most preferably thebacterium is Staphylococcus aureus.

It is also envisaged by the present invention that the inhibitors can becombined with each other. As such in one embodiment the MEK inhibitor iscombined with another MEK inhibitor, the p38 inhibitor and/or the NFκBinhibitor. In further embodiments, the p38 inhibitor is combined withanother p38 inhibitor, the MEK inhibitor and/or the NFκB inhibitor. Inanother embodiment the NFκB inhibitor is combined with another NFκBinhibitor, the p38 inhibitor and/or the MEK inhibitor. With respect tothe above, the term “another inhibitor” is used to clarify that e.g. oneMEK inhibitor can also be combined with another MEK inhibitor, whilethese two MEK inhibitors are not the same. E.g. the MEK inhibitorCI-1040 can be combined with the MEK inhibitor GDC-0973. This equallyrelates to the p38 and NFκB inhibitors.

In one embodiment, the MEK inhibitor, the p38 inhibitor and/or the NFκBinhibitor is/are administered contemporaneously, previously orsubsequently to the one or more additional inhibitors targeting theinfluenza virus and the bacterium.

In further embodiments, the MEK inhibitor, p38 inhibitor and/or NFκBinhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection of the present invention, wherein the MEKinhibitor, the p38 inhibitor and/or the NFκB inhibitor are combined withone or more inhibitors targeting the influenza virus and/or thebacterium. In one embodiment, the MEK inhibitor, the p38 inhibitorand/or the NFκB inhibitor is/are administered contemporaneously,previously or subsequently to the one or more inhibitors targeting theinfluenza virus and/or the bacterium.

In general, an inhibitor targeting the influenza virus is any inhibitoror medicament effective in influenza therapy. Different substances areknown to be effective in reducing an influenza infection. Among them arefor example neuraminidase inhibitors, compounds targeting an ion channelprotein (M2) and compounds targeting polymerase or endonuclease activityvia interfering with a component of the viral polymerase complex, PB1,PB2, PA or NP. By the invention also pharmaceutically acceptable saltsof these inhibitors are envisioned.

A “neuraminidase inhibitor” is an antiviral drug targeted at influenzavirus, which works by blocking the function of the viral neuraminidaseprotein, thus preventing the virus from binding to a cell it aims toinfect and/or preventing the virus from reproducing by budding from thehost cell, since the newly produced viruses cannot bud off from the cellin which they have replicated. Also comprised are pharmaceuticallyacceptable salts of a neuraminidase inhibitor. Preferred neuraminidaseinhibitors are oseltamivir, zanamivir, peramivir, or a pharmaceuticallyacceptable salt of any of these substances, such as oseltamivirphosphate, oseltamivir carboxylate, etc. Most preferred neuraminidaseinhibitors are oseltamivir phosphate, zanamivir, oseltamivir orperamivir.

Compounds targeting an ion channel protein (M2) are for exampleamantadine and/or rimantadine, while compounds targeting polymerase orendonuclease activity via interfering with a component of the viralpolymerase complex, PB1, PB2, PA or NP are for example the NP blockerNucleozin or the polymerase inhibitor T-705.

Alternatively or additionally, the MEK inhibitor, p38 inhibitor and/orNFκB inhibitor can be combined with one or more inhibitors targeting thebacterium. An inhibitor targeting the bacterium can be any inhibitoreffective in reducing bacterial infection. A preferred inhibitor, wellknown to the skilled artesian is an antibiotic. Preferred antibioticscan be obtained from table 5 (FIG. 11). Thus, in one embodiment, theantibiotic is selected from the group consisting of the antibiotics aslisted in table 5 (FIG. 11). In a further embodiment, the antibiotic isselected from the group consisting of the class of antibiotics as listedin table 5 (FIG. 11). In another embodiment, the antibiotic is selectedfrom the group consisting of the generic name of the antibiotics aslisted in table 5 (FIG. 11). More preferred are antibiotics selectedfrom Gentamicin, Rifampicin, Lysosthaphin, Erythromycin, Levofloxacin,Vancomycin, Teicoplanin, Penicillin and Oxacillin.

The “subject”, which may be treated by the inhibitors or combinations ofinhibitors of the present invention preferably, is a vertebrate. In thecontext of the present invention the term “subject” means an individualin need of a treatment of a co-infection or a bacterial infection alone.Preferably, the subject is a patient suffering from a co-infection or abacterial infection alone or being at a risk thereof. Preferably, thepatient is a vertebrate, more preferably a mammal. Mammals include, butare not limited to, farm animals, sport animals, pets, primates, miceand rats. Preferably, a mammal is as a human, dog, cat, cow, pig, mouse,rat etc., particularly preferred, it is a human. In some embodiments,the subject is a human subject, which optionally is more than 1 year oldand less than 14 years old; between the ages of 50 and 65, or older than65 years of age. In other embodiments the the subject is a humansubject, which is selected from the group consisting of subjects who areat least 50 years old, subjects who reside in chronic care facilities,subjects who have chronic disorders of the pulmonary or cardiovascularsystem, subjects who required regular medical follow-up orhospitalization during the preceding year because of chronic metabolicdiseases, renal dysfunction, hemoglobinopathies, or immunosuppression,subjects with less than 14 years of age, subjects between 6 months and18 years of age who are receiving long-term aspirin therapy, and womenwho will be in the second or third trimester of pregnancy during theinfluenza season.

In the method of the invention, the MEK inhibitor, p38 inhibitor or NFκBinhibitor as well as the inhibitor targeting the influenza virus and theinhibitor targeting the bacterium may be administered orally,intravenously, intrapleurally, intramuscularly, topically or viainhalation. Preferably, the MEK inhibitor is administered via nasalinhalation or orally.

The present invention also envisages different compositions. The presentinvention relates to a composition comprising a MFK inhibitor, a p38inhibitor and/or a NFκB inhibitor for use in a method for theprophylaxis and/or treatment of a co-infection comprising a bacterialinfection and an influenza virus infection. The present inventionsimilarly relates composition comprising a MEK inhibitor, a p38inhibitor and/or a NFκB inhibitor for use in a method for theprophylaxis and/or treatment of a bacterial infection. Also provided forby the present invention is a composition comprising a MEK inhibitor, ap38 inhibitor and/or a NFκB inhibitor and one or more inhibitorstargeting the influenza virus and/or the bacterium for use in a methodfor the prophylaxis and/or treatment of a co-infection comprising abacterial infection and an influenza virus infection. In addition, thepresent invention relates to a composition comprising a MEK inhibitor, ap38 inhibitor and/or a NFκB inhibitor and one or more inhibitorstargeting the the bacterium for use in a method for the prophylaxisand/or treatment of a bacterial infection.

The composition comprising the MEK inhibitor, the p38 inhibitor and/orthe NFκB inhibitor and additionally eventually one or more inhibitorstargeting the the bacterium and/or one or more inhibitors targeting theinfluenza virus may be a pharmaceutical composition. Preferably, suchcompositions further comprise a carrier, preferably a pharmaceuticallyacceptable carrier. The composition can be in the form of orallyadministrable suspensions or tablets; nasal sprays, sterile injectablepreparations (intravenously, intrapleurally, intramuscularly), forexample, as sterile injectable aqueous or oleaginous suspensions orsuppositories. When administered orally as a suspension, thesecompositions are prepared according to techniques available in the artof pharmaceutical formulation and may contain microcrystalline cellulosefor imparting bulk, alginic acid or sodium alginate as a suspendingagent, methylcellulose as a viscosity enhancer, and sweeteners/flavoringagents known in the art. As immediate release tablets, thesecompositions may contain microcrystalline cellulose, dicalciumphosphate, starch, magnesium stearate and lactose and/or otherexcipients, binders, extenders, disintegrants, diluents, and lubricantsknown in the art. The injectable solutions or suspensions may beformulated according to known art, using suitable non-toxic,parenterally acceptable diluents or solvents, such as mannitol,1,3-butanediol, water, Ringer's solution or isotonic sodium chloridesolution, or suitable dispersing or wetting and suspending agents, suchas sterile, bland, fixed oils, including synthetic mono- ordiglycerides, and fatty acids, including oleic acid.

The inhibitor or inhibitors are preferably administered in atherapeutically effective amount.

The pharmaceutical composition for the use of the invention andcomprising a MEK inhibitor, a p38 inhibitor and/or a NFκB inhibitor andoptionally one or more inhibitors targeting an influenza virus and/orone or more inhibitors targeting a bacterium is administered to apatient which is a mammal or a bird. Examples of suitable mammalsinclude, but are not limited to, a mouse, a rat, a cow, a goat, a sheep,a pig, a dog, a cat, a horse, a guinea pig, a canine, a hamster, a mink,a seal, a whale, a camel, a chimpanzee, a rhesus monkey and a human,with human being preferred. Examples of suitable birds include, but arenot limited to, a turkey, a chicken, a goose, a duck, a teal, a mallard,a starling, a Northern pintail, a gull, a swan, a Guinea fowl or waterfowl to name a few. Human patients are a particular embodiment of thepresent invention.

The “therapeutically effective amount” for each activecompound/inhibitor can vary with factors including but not limited tothe activity of the compound used, stability of the active compound inthe patient's body, the severity of the conditions to be alleviated, thetotal weight of the patient treated, the route of administration, theease of absorption, distribution, and excretion of the active compoundby the body, the age and sensitivity of the patient to be treated,adverse events, and the like, as will be apparent to a skilled artisan.The amount of administration can be adjusted as the various factorschange over time.

The inhibitors, methods and uses described herein are applicable to bothhuman therapy and veterinary applications. The compounds describedherein, in particular, MEK inhibitor, a p38 inhibitor and/or a NFκBinhibitor and optionally one or more inhibitors targeting an influenzavirus and/or one or more inhibitors targeting a bacterium having thedesired therapeutic activity may be administered in a physiologicallyacceptable carrier to a subject, as described herein. Depending upon themanner of introduction, the compounds may be formulated in a variety ofways as discussed below. The concentration of therapeutically activecompound in the formulation may vary from about 0.1-100 wt %. The agentsmaybe administered alone or in combination with other treatments.

The pharmaceutical compounds in the method of present invention can beadministered in any suitable unit dosage forms. Suitable oralformulations can be in the form of tablets, capsules, suspension, syrup,chewing gum, wafer, elixir, and the like. Pharmaceutically acceptablecarriers such as binders, excipients, lubricants, and sweetening orflavoring agents can be included in the oral pharmaceuticalcompositions. If desired, conventional agents for modifying tastes,colors, and shapes of the special forms can also be included.

For injectable formulations, the pharmaceutical compositions can be inlyophilized powder in admixture with suitable excipients in a suitablevial or tube. Before use in the clinic, the drugs may be reconstitutedby dissolving the lyophilized powder in a suitable solvent system toform a composition suitable for intravenous or intramuscular injection.

In accordance with another embodiment of the present invention, apharmaceutical composition is provided, comprising a therapeuticallyeffective amount of a a MEK inhibitor, a p38 inhibitor and/or a NFκBinhibitor as well as a therapeutically effective amount of aneuraminidase inhibitor chosen from the group of oseltamivir,oseltamivir phosphate, zenamivir and peramivir.

In one embodiment, the composition can be in an orally administrableform (e.g., tablet or capsule or syrup etc.) with a therapeuticallyeffective amount (e.g., from 0.1 mg to 2000 mg, 0.1 mg to 1000 mg, 0.1to 500 mg, 0.1 to 200 mg, 30 to 300 mg, 0.1 to 75 mg, 0.1 to 30 mg) of aMEK inhibitor, a p38 inhibitor and/or a NFκB inhibitor and atherapeutically effective amount (e.g., from 0.1 mg to 2000 mg, 0.1 mgto 1000 mg, 0.1 to 500 mg, 0.1 to 200 mg, 30 to 300 mg, 0.1 to 75 mg,0.1 to 30 mg) of neuraminidase inhibitor as described above.

In further embodiments, the MEK inhibitor, p38 inhibitor and/or NFκBInhibitor is/are for use in the methods for the prophylaxis and/ortreatment of a co-infection of the present invention, wherein the MEKinhibitor, the p38 inhibitor and/or the NFκB inhibitor reduces both theviral and bacterial infection, when contacting it/them with an in vitrotest system, wherein the test system comprises cultured cells infectedwith

-   -   a) an influenza virus and    -   b) a bacterium        when compared to the in vitro test system before the contacting.        In another embodiment, the MEK inhibitor, p38 inhibitor and/or        NFκB inhibitor is/are for use in the methods for the prophylaxis        and/or treatment of a bacterial infection of the present        invention, wherein the MEK inhibitor, the p38 inhibitor and/or        the NFκB inhibitor reduces the bacterial infection, when        contacting it/them with an in vitro test system, wherein the        test system comprises cultured cells infected with a bacterium,        when compared to the in vitro test system before the contacting.

As such the present invention also relates to an in vitro test system,wherein the test system comprises cultured cells infected with

-   -   a) an influenza virus and    -   b) a bacterium.        Along this line, the present invention also provides for an in        vitro test system, wherein the in vitro test system comprises        cultured cells infected with a bacterium.

In the cases where the in vitro test system includes a viral andbacterial infection, again, these infections can be taking placesequentially or simultaneously.

A “cultured cell” or “cultured cells” is/are cells, which are notpresent in their natural environment e.g. within a plant or animal.Rather a cultured cell may be a primary cell culture, which comprisescells isolated from their natural environment, or a cell line.Preferably the cultured cells are human lung epithelial cells.Preferably, the cultured cells are seeded at a density of about 1×10⁵,2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 10×10⁵, 11×10⁵most preferably 8×10⁵ cells in 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml,3.5 ml, 4 ml medium such as DMEM. Most preferred is a density of 8×10⁵cells per in 2 ml DMEM.

Such cultured cells are infected with a virus and a bacterium or inother embodiments with a bacterium alone. As already described above, aco-infection can take place in a sequential or simultaneous manner. Forexample the cultured cells may be infected first with the influenzavirus and 30 minutes later with bacterium/bacteria. It is also possibleto additionally add an antibiotic to the culture after 3 hours, toremove extracellular bacteria. In such a scenario the antibioticum wouldthen become washed off again. In other embodiments, the cells are onlyinfected with a bacterium.

The term “contacting” as used herein refers to the bringing of a cellcomprising an influenza virus and a bacterium spatially into closeproximity to a MEK inhibitor, a p38 inhibitor and/or a NFκB inhibitor.

This can for example mean that an inhibitor is applied to the medium inwhich the cultured cells are located via a syringe.

Upon contacting then, if the inhibitor is active, the viral infection aswell as the bacterial infection becomes reduced. In some embodiments,again, the inhibitor of the present invention is used to reduce only abacterial infection in the absence of an influence virus infection.

In one embodiment, the reduction of the viral infection is a reductionin plaque forming units (pfu)/ml and the reduction in the bacterialinfection is a reduction in colony forming units (CFU)/ml. The “plaqueforming units (pfu)/ml” is a measure of the number of particles capableof forming plaques per unit volume, such as virus particles. It is afunctional measurement rather than a measurement of the absolutequantity of particles: viral particles that are defective or which failto infect their target cell will not produce a plaque and thus will notbe counted. For example, a solution of influenza virus with aconcentration of 1,000 PFU/μl indicates that 1 μl of the solutioncontains enough virus particles to produce 1000 infectious plaques in acell monolayer. In the case of the present invention, a cell culturetreated with an inhibitor shows a reduced number of plaque forming unitsin a culture after the treatment, when compared to a culture before thetreatment with an inhibitor of the present invention.

A possible “reduction in plaque forming units (pfu)/ml” is analysed inthe following way. First the cultured cells, which are co-infected withan influenza virus and a bacterium are analysed for their ability togenerate plaque forming units (pfu)/ml by e.g. sucking of some cellsfrom the petridish and plating them for counting the bacterial plaquesthat will form. This result is then compared to the number of plaqueforming units (pfu)/ml generated by cells of the same culture after theinhibitor was applied. If the number of the plaque forming units(pfu)/ml is reduced after the treatment with an inhibitor compared tothe number generated before the application of the inhibitor, there is areduction in the plaque forming units.

The “colony forming units (CFU)/ml” estimates the number of viablebacteria in a sample. Different methods exist. For example to generatecolony forming units a sample (e.g. cultured cells in a small volume)are spread across the surface of a nutrient agar plate and allowed todry before incubation for counting. A viable bacterium is defined as theability to multiply via binary fission under the controlled conditions.The visual appearance of a colony in a cell culture requires significantgrowth—when counting colonies it is uncertain if the colony arose fromone cell or 1,000 cells. Therefore results are reported as CFU/ml(colony-forming units per milliliter) for liquids, and CFU/g(colony-forming units per gram) for solids to reflect this uncertainty(rather than cells/ml or cells/g).

“Colony forming units (CFU)/ml” can be analysed in the following way.First the cultured cells, which are co-infected with an influenza virusand a bacterium or with a bacterium alone are analysed for their abilityto generate colony forming units (CFU)/ml by e.g. sucking of some cellsfrom the petridish and plating them for counting. This result is thencompared to the number of colony forming units (CFU)/ml generated bycells of the same culture after the inhibitor was applied. If the numberof the colony forming units (CFU)/ml is reduced to the number generatedbefore the application of the inhibitor, there is a reduction.

In general the person skilled in the art knows these well knowntechniques of analyzing bacterial and viral infections. How one canmeasure the plaque forming units (pfu)/ml and the colony forming units(CFU)/ml is further described in the literature (Tuchscherr, L. et al.(2011). Staphylococcus aureus phenotype switching: an effectivebacterial strategy to escape host immune response and establish achronic infection (EMBO molecular medicine 3, 129-141 and Hrincius, E. Ret al. (2010) CRK adaptor protein expression is required for efficientreplication of avian influenza A viruses and controls JNK mediatedapoptotic responses. Cellular microbiology 12, 831-843).

In addition the present invention relates to the following items:

Item 1. The invention also provides for the use of the in vitro testsystem of of the present invention for the determination of inhibitorseffective in reducing a coinfection comprising a bacterial infection andan influenza virus infection. In one embodiment, the reduction of theviral infection is a reduction in plaque forming units (pfu)/ml and thereduction in the bacterial infection is a reduction in colony formingunits (CFU)/ml.

Item 2. In addition the present invention relates to a method fordetecting molecules effective in the prophylaxis and/or treatment of aco-infection comprising a bacterial infection and an influenza virusinfection comprising contacting the in vitro test system of the presentinvention with a compound of interest, wherein the compound of interestreduces both the viral and bacterial infection, compared to the in vitrotest system before the contacting. In one embodiment, the reduction ofthe viral infection is a reduction in plaque forming units (pfu)/ml andthe reduction in the bacterial infection is a reduction in colonyforming units (CFU)/ml.

Item 3. The present invention, in addition, relates to a use of the invitro test system of the present invention for the determination ofinhibitors effective in reducing a bacterial infection.

Item 4. Furthermore, the present invention relates to the use of the invitro test systems of the present invention for the examination ofinnate host cell responses, which optionally includes examination of thelevel of signal transduction, resulting cytokine and chemokineexpression, induction of apoptosis and necrosis and/or redox hemostasisregulating health and disease.

Item 5. Also provided for by the present invention is a method fordetecting molecules effective in the prophylaxis and/or treatment abacterial infection comprising contacting the in vitro test system ofthe present invention with a compound of interest, wherein the compoundof interest reduces the bacterial infection, compared to the in vitrotest system before the contacting.

Item 6. The present invention furthermore relates to a cultured cellinfected with an influenza virus and a bacterium.

Item 7. Also provided for is a cultured cell infected with a bacterium.

Item 8. The present invention also relates to a method for theprophylaxis and/or treatment of a co-infection comprising a bacterialinfection and an influenza virus infection in a subject, comprisingadministering a therapeutically effective amount of a MEK inhibitor, ap38 inhibitor and/or a NFκB inhibitor of the present invention or apharmaceutical composition of the present invention to said subject.

Item 9. Also the present invention provides for a use of a MEKinhibitor, a p38 inhibitor and/or a NFκB inhibitor of the presentinvention or a composition of the present invention for the preparationof a medicament.

Item 10. In addition the present invention relates to a use of a MEKinhibitor, a p38 inhibitor and/or a NFκB inhibitor of the presentinvention or a composition of the present invention for the prophylaxisand/or treatment of a co-infection comprising a bacterial infection andan influenza virus infection.

Item 11. Similarly, the present invention also provides for a method forthe prophylaxis and/or treatment of a bacterial infection in a subject,comprising administering a therapeutically effective amount of a MEKinhibitor, a p38 inhibitor and/or a NFκB inhibitor of the presentinvention or a pharmaceutical composition of the present invention tosaid subject.

Item 12. In addition the present invention relates to a use of a MEKinhibitor, a p38 inhibitor and/or a NFκB inhibitor of the presentinvention or a composition of the present invention for the prophylaxisand/or treatment of a bacterial infection.

It must be noted that as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

All publications and patents cited in this disclosure are incorporatedby reference in their entirety. To the extent the material incorporatedby reference contradicts or is inconsistent with this specification, thespecification will supersede any such material.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or sometimes when used herein with the term “having”.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” may be replaced with either of theother two terms.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

EXAMPLES

The following examples illustrate the invention. These examples shouldnot be construed as to limit the scope of this invention. The examplesare included for purposes of illustration and the present invention islimited only by the claims.

Example 1

Within the last years the need for additional and alternativetherapeutic strategies apart from vaccination or treatment withconventional antivirals against IV (neuraminidase and M2 blockers) andconventional antibiotics against S. aureus steadily increased. Inmeantime for antiviral intervention several cellular factors have beenidentified as potential targets. In quite contrast, the knowledge aboutthe rote of cellular factors during bacterial infection and inparticular as targets for antibacterial treatment either by reduction ofbacterial amount and/or onset of accelerating cytokine expression isless understood, even more in presence of IV co-infection.

We established an infection protocol that allows determination of (1)progeny virus titers and (2) titers of intracellular bacteria as well as(3) changes in host defense mechanisms in presence or absence ofpotential antiinfectives upon IV and S. aureus co-infection. In aninitial approach we investigated the effect of the MEK inhibitor(U0126=50 μM), the p38 inhibitor (SB202190=10μM) and the NFκB inhibitor(LASAG=5 mM) in comparison to a solvent control against IV and S. aureusinfection in a singular or co-infection situation. As a control theviral neuraminidase inhibitor Oseltamivir (Tamiflu) (2 μm) was used incomparison to Hepes. For virus infection we used the human influenzavirus A/Puerto Rico/8134 (H1N1) or the avian influenza virusA/FPV/Bratislava/79 (H7N7) and for bacterial infection we used the S.aureus strain 6850. Procedure of infection (FIG. 1):

Human lung epithelial cells were seeded in 6-well plates (8×10⁵cells/well) in 2 ml DMEM [10% FCS]. 16-20 hrs after seeding, cells wererinsed and incubated with PBS/BA [0.2% bovine serum albumin (BSA), 1 mMMgCl₂, 0.9 mM CaCl₂, 100 U/mI penicillin, 0.1 mg/ml streptomycin] (500μl per 6 well) or PBS/BA containing the virus at the indicatedmultiplicity of infection (MOI) at 37° C. After 30 min incubation, thevirus dilution was aspirated, cells were rinsed with PBS andsupplemented with invasion medium DMEM/INV [1% human serum albumin, 25nmol/l HEPES] (2 ml per 6 well) with or without bacteria at the MOIindicated in presence or absence of the test compound. 3 hrs postbacterial infection cells were treated with antibiotics to removeextracellular bacteria. Therefore cells were rinsed with PBS andsubsequently incubated with DMEM/INVantibiotics [2 μg/ml lysostaphin(Sigma)] (1 ml per 6 well) for 20 min at 37° C.. After an additionalwash with PBS cells were supplemented with DMEM/INV containing the testsubstance and were incubated for the times indicated at 37° C. UponA/Puerto Rico/8/34 DMEM/INV was additionally supplemented with 0.333μg/ml Trypsin (Invitrogen). Determination of IV titers and intracellularbacteria were performed as described in (Hrincius et al., 2010,Tuchscherr et at., 2011).

IV titers are depicted as plaque forming units (pfu)/ml and S. aureustiters are depicted as colonie forming untits (CFU)/ml. Data representthe means±SD of two to three independent experiments with two biologicalsamples. Statistical significance was evaluated by a two-tailed twosample t-test (*p<0.05; **p<0.01; ***p<0001).

In a co-infection situation the presence of S. aureus affected IVreplication and the presence of IV affected the intracellular amount ofS. aureus levels, respectively, due to changes in innate immuneresponses as well as autophagic and apoptotic mechanisms. Nonetheless,as expected we observed inhibitory effects of U0126 (FIG. 2, 3),SB202190 (FIG. 4) and LG-ASA (FIG. 5) on IV replication. Interestingly,viral titers were also reduced upon treatment with these inhibitors inpresence of S. aureus. Furthermore, we were agreeably surprised whenintracellular amounts of S. aureus were reduced in presence of U0126,SB202190 or LG-ASA independent from the absence or presence of IV.Another, amazing observation concerned bacterial replication in presenceof U0126 (50μM). When S. aureus was cultivated over night at 37° C. inDMEM/INV without living cells, bacterial titers were already reduced,but to the same degree than during infection of cells, indicating thedependence of bacteria from a cellular factor (FIG. 2E).

As control we investigated viral titers and intracellular bacterialamounts upon application of the viral neuraminidase inhibitorOseltamivir (Tamiflu) (FIG. 6). While IV titers were significantlyreduced in absence or presence of S. aureus, intracellular bacterialamounts were rather increased.

The results demonstrate the great potential of substances targetingcellular factors as antiinfectives against IV and S. aureusco-infection, rather than substances against the pathogen itself.

Example 2

In further experiments the effect of the MEK inhibitor U0126, CI-1040and Cobimetinib (GDC-0973) in comparison to a solvent control againstinfluenza A virus (IAV) and S. aureus 6850 infection in a singular orco-infection situation were investigated.

The co-infection procedure is depicted in FIG. 12. To examine theviability of human lung epithelial cells in our experimental setting,cell morphology was monitored 18 hrs upon infection by light microscopy(FIG. 13), the time at which the pathogen load was determined (FIG. 14).

As visible in FIG. 13 singular infection with S. aureus 6850 (6850), theinfluenza virus strains A/FPV/Bratislava/79 (H7N7) (FPV) orA/Wisconsin/67/2005 (H3N2), as well as the co-infection resulted inslight but clearly detectable cell damage. In presence of U0126 (50 μM)the cell layer appeared much less damaged.

The effect of the MEK inhibitor U0126 (50 μM) against influenza virusreplication A/FPV/Bratislava/79 (H7N7) (FPV) or A/Wisconsin/67/2005(H3N2) in comparison to a solvent control was determined in human lungepithelial cells in a singular or co-infection situation (FIG. 14).

Inhibition of MEK/ERK signalling resulted in a significant reduction ofvirus titers upon infection with the IAV subtypes H7N7 and H3N2 in asingular infection situation (FIG. 14). Virus titers were reduced in aco-infection situation in the presence of U0126, too, up to significantlevels in H7N7/S. aureus co-infected cells (FIG. 14).

Furthermore, the effect of the MEK inhibitor U0126 (50 μM) againstinternalized S. aureus 6850 was analysed (FIG. 15A, C). In thisexperimental setting bacterial titers were only slightly decreased inpresence of the inhibitor.

To further investigate the effect of U0126 on bacterial growth ingeneral, a cell-free over-night culture of S. aureus 6850 wassupplemented with different amounts of U0126 (10 μM and 50 μM) orsolvent (FIG. 15B, D). Bacterial growth was inhibited in presence ofU0126 in a concentration dependent manner, in comparison to the solventcontrol (FIG. 15B, D).

Since pro-inflammatory cytokine- and chemokine expression contributes tosevere inflammation and tissue damage, the mRNA synthesis of respectivechemokines, such as CCL3, also known as macrophage inflammatory protein1α (MIP1α) and CCL5, also known as RANTES were analysed by qRT-PCR in aninfection experiment in presence or absence of U0126 (50 μM) (FIG. 16A,B). The IAV-induced CCL3 mRNA synthesis, which was increased in presenceof S. aureus 6850, was reduced in presence of U0126. Similarly, theIAV-induced CCL5 mRNA synthesis, which was reduced in presence of S.aureus, was further reduced in presence of U0126 (50 μM).

In western-blot analysis the inhibitory effect of U0126 on MEK/ERKsignalling was verified by use of a phosphospecific ERK1/2 antibody(FIG. 16C). Furthermore, a reduction of viral protein synthesis (PB1)was observed in presence of upon inhibition of MEK/ERK signalling.

To verify the anti-pathogen potential of U0126 in an in vivo mousemodel, influenza virus-infected mice were left untreated or treated withU0126 daily and superinfected with S. aureus 6850 (FIG. 17). Theadministration of U0126 led to a reduction in bacterial titers in vivoindependent of viral titers. The fail of reduced virus titers might beexplained by the late administration of U0126 at a time point when virustiters are already decreasing in the infection course. Formerexperiments have indicated that the inhibitor has a higher inhibitoryeffect, when it is given before influenza virus infection. Nonetheless,the bacterial titers were significantly reduced upon application ofU0126.

Within other approaches the effect of the MEK inhibitor CI-1040 (10 μM)against influenza virus replication A/FPV/Bratislava/79 (H7N7) (FPV) orA/Puerto Rico/8/34 (H1N1) in comparison to a solvent control wasdetermined in human lung epithelial cells in a singular or co-infectionsituation (FIG. 18).

Inhibition of MEK/ERK signalling by CI-1040 resulted in a reduction ofvirus titers upon infection with the IAV subtypes H7N7 and H1N1 in asingular and co-infection situation (FIG. 18).

To further investigate the effect of CI-1040 on bacteria growth ingeneral, a cell-free over-night culture of S. aureus 6850 wassupplemented with different amounts of CI-1040 (1 μM and 10 μM) orsolvent (FIG. 19A, B). Bacterial growth was slightly inhibited inpresence of CI-1040 in a concentration dependent manner, in comparisonto the solvent control (FIG. 19A, B).

To verify the anti-pathogen potential of another MEK inhibitor,Cobimetinib was tested in an in vivo mouse model, influenzavirus-infected mice were left untreated or treated daily withCobimetinib and were super-infected with S. aureus 6850 (FIG. 20). Theadministration of Cobimetinib led to a slight but clearly detectablereduction in viral and bacterial titers in vivo. Since it has been shownrecently that the maximal tolerated dose of Cobimetinib is 30 mg/kg/day.Thus, the inhibitory effect might be improved by higher dosages thanused in the present experiment (10 mg/kg/day), which was far less fromthe maximal tolerated dosage.

In conclusion, the results show different MEK inhibitors as potentialanti-IAV/S. aureus substances.

Example 3

In further experiments the effect of the NFκB-inhibitor LG-ASA (LASAG)against influenza A virus (IAV) and S. aureus 6850 infection in asingular or co-infection situation were investigated.

The co-infection procedure is depicted in FIG. 21 (upper part). Toexamine the viability of human lung epithelial cells upon infection inabsence and presence of LG-ASA (5 mM) cell morphology was monitored 18hrs upon infection by light microscopy (FIG. 21). While infection withIAV and/or S. aureus in absence of LG-ASA (left panel) results in celldestruction, cell morphology was improved in presence of LG-ASA (rightpanel).

The effect of the NFκB-inhibitor LG-ASA (5 mM) against influenza virusreplication A/Puerto Rico/8/34 (H1N1) was determined in human lungepithelial cells in a singular or co-infection situation (FIG. 22/23) 8h (FIGS. 22/23 A, B, E, F) and 18 h (FIG. 22/23 C, D, G, H) postinfection. Two different S. aureus strains were used for infection (a)S. aureus SH1000 (FIG. 22/23 A-D) and (b) S. aureus 6850 (FIG. 22/23E-H).

While IAV replication was reduced in presence of LG-ASA 8 h and 18 hupon infection, a reduction of bacterial titers was only visible 18 hp.i. In FIG. 22 the results are depicted in a linear scale. To bettervisualize the pathogen inhibitory effect of LG-ASA, the untreatedcontrols of the three independent experiments were arbitrarily set as100% and the mean is presented (FIG. 23).

Since LG-ASA was added directly during bacterial infection, theseresults indicate a very early effect on bacterial internalization, whichpotentiates during ongoing release and new bacterial internalization.

Very recently it has been shown that NFκB is required for phagocytosisof S. aureus by monocytes (Zhu et al., 2014; Exp. Cell Res. 1;327(2):256-63). Based on these findings and our own observations wewanted to know if LG-ASA prevents bacterial uptake. We pre-treated humanlung epithelial cells 4 hours before bacterial infection and determinedinternalized bacteria up to two hours post infection. To ensure thatonly internalized bacteria were detected, non-internalized bacteria wereremoved by an antibiotic wash prior to cell lysis. The data indicate atime dependent uptake of S. aureus 6850, which is blocked in presence ofLG-ASA (FIG. 24A, B). A concentration dependent inhibitory effect ofLG-ASA could be further demonstrated on the bacterial strain S. aureusUSA300 (FIG. 24C, D). To better visualize the pathogen inhibitory effectof LG-ASA, which is visible in FIG. 24A, C the untreated controls of thethree independent experiments were arbitrarily set as 100% and the meanis depicted (FIG. 24B, D).

To verify the importance of NFκB-mediated signalling onto S. aureusinternalisation, NFκB was induced by TNF-α stimulation 4 hours priorbacterial infection. The activation of NFκB resulted in the enhanceduptake of S. aureus 6850 and S. aureus USA300. As a controlTNF-α-induced activation was simultaneously blocked by LG-ASA, whichresulted in the inhibition of TNF-α-promoted bacterial uptake, asexpected (FIG. 25).

To verify the anti-pathogen potential of LG-ASA in an in vivo mousemodel different co-infection settings in presence and absence of LG-ASAwere tested.

As visible in FIG. 26 treatment of IAV/S. aureus co-infected mice withLG-ASA results in enhanced survival (FIG. 26A) and reduced body weightloss (FIG. 26B).

In conclusion, our results show that NFκB inhibitors such as LG-ASA actas anti-IAV/S. aureus substances in vitro and in vivo.

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What is claimed is:
 1. A method of treating a respiratory co-infectioncomprising a bacterial infection and an influenza virus infection in ahuman patient comprising administering to the patient an effectiveamount of a p38 inhibitor, wherein the bacterial infection is by aStaphylococcaceae bacterium or a Streptococcaceae bacterium.
 2. Themethod of claim 1, wherein the bacterial infection is by aStaphylococcaceae aureus bacterium.
 3. The method of claim 1, whereinthe influenza virus infection is by an influenza A virus or an influenzaB virus.
 4. The method of claim 3, wherein the influenza A virus isH1N1, H2N2, H3N2, H6N1, H7N7, H7N9, H9N2 H10N7, H10N8 or H5N1.
 5. Themethod of claim 1, wherein the p38 inhibitor is selected from the groupconsisting of SB202190, LY2228820, CAY10571, SB 203580, Tie2 KinaseInhibitor,2-(4-Chlorophenyl)-4-(fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one,CGH 2466, SB220025, Antibiotic LL Z1640-2, TAK 715, SB202190hydrochloride, SKF 86002, AMG548, CMPD-1, EO 1428, JX 401, ML 3403, RWJ67657, SB 202190, SB 203580, SB 203580 hydrochloride, SB 239063, SCIO469, SX 011, TAK 715, Pamapimod, Losmapimod (GW856553), Dilmapimod(SB681323), VX 702, VX 745, Doramapimod (BIRB 796), BMS-582949,ARRY-797, PH797804 preferably VX-702, SB202190, Pamapimod, Losmapimod(GW856553), Dilmapimod (SB681323), Doramapimod (BIRB 796), BMS-582949,ARRY-797, PH797804 and SCIO-469.
 6. The method of claim 1, wherein thep38 inhibitor is combined with another p38 inhibitor inhibitor, a MEKinhibitor, an NFκB inhibitor, or combinations thereof.
 7. The method ofclaim 1, wherein the p38 inhibitor is combined with one or moreinhibitors targeting the influenza virus, the bacterium, or both thevirus and the bacterium.
 8. The method of claim 7, wherein the p38inhibitor is administered contemporaneously, previously or subsequentlyto the one or more inhibitors targeting the influenza virus, thebacterium, or both the virus and the bacterium.
 9. The method of claim7, wherein the one or more inhibitors targeting the influenza virus is aneuraminidase inhibitor.
 10. The method of claim 9, wherein theneuraminidase inhibitor is oseltamivir phosphate, zanamivir,oseltamivir, or peramivir
 11. The method of claim 7, wherein the one ormore inhibitors targeting the influenza virus is a compound targeting anion channel protein (M2).
 12. The method of claim 11, wherein thecompound targeting ion channel protein (M2) is amantadine, rimantadine,or both amantadine and rimantadine.
 13. The method of claim 7, whereinthe one or more inhibitors targeting the influenza virus is a compoundtargeting polymerase or endonuclease activity via interfering with acomponent of the viral polymerase complex, PB1, PB2, PA, or NP.
 14. Themethod of claim 13, wherein the compound targeting polymerase orendonuclease activity via interfering with a component of the viralpolymerase complex, PB1 PB2, PA, or NP is NP blocker Nucleozin orpolymerase inhibitor T-705.
 15. The method of claim 7, wherein the oneor more inhibitors targeting the bacterium is an antibiotic.
 18. Themethod of claim 15, wherein the antibiotic is Gentamicin, Rifampicin,Lysosthaphin, Erythromycin, Levofloxacin Vancomycin, Teicoplanin,Penicillin, or Oxacillin.